TW202405023A - Compositions and methods for treatment of cancer - Google Patents

Abstract

Fusion proteins and their use in treating subjects having cancer are described.

Description

Compositions and methods for treating cancer

This invention describes fusion proteins and their use in treating individuals with cancer.

The past decade has witnessed a transformation in cancer treatment for relapsed and refractory B malignancies. The use of CD19-targeted cell therapies may provide high clinical response rates in some of these otherwise difficult-to-treat patient populations. Additionally, some patients achieved durable clinical remission that appeared to be curative. However, these results are tempered by the finding that about half of the patients who initially responded to cell therapy relapsed, and most relapsed quickly, usually within six months. Therefore, additional therapeutic agents are needed for the treatment of cancer.

In some embodiments, the invention provides a fusion protein comprising (i) an antibody or antigen-binding fragment thereof that binds a tumor antigen; and (ii) a polypeptide of interest. In one aspect, the invention provides a fusion protein comprising (i) an antibody that binds a tumor antigen or an antigen-binding fragment thereof; (ii) a polypeptide of interest; and (iii) a half-life extending polypeptide. In some embodiments, the fusion protein includes at least a first linker. In some embodiments, the fusion protein includes at least a first linker and a second linker.

In some embodiments, the half-life extending polypeptide is any of the following: hyaluronic acid binding motif, PAS polypeptide, proline/alanine random coil polypeptide and albumin or fragment (eg, HSA), albumin binding polypeptide and antibodies or antigen-binding fragments thereof. In some embodiments, the half-life extending polypeptide is an anti-albumin antibody or antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment thereof is an anti-CD20 antibody or antigen-binding fragment thereof. In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof comprises an anti-CD20 VHH. In some embodiments, the anti-CD20 VHH comprises the amino acid sequence of any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53.

In some embodiments, the polypeptide of interest is a B cell antigen. In some embodiments, the polypeptide of interest is CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, CD180, ROR1, BCMA, HLA-DR10, CD1, CD5, CD21, CD25, CD27, CD30, CD38, CD78 , any of CD80, CD86, CD138, CD319, surface Ig, PD-1, PD-L1, PD-L2, TGFbR2, CD79a and CD79b. In some embodiments, the polypeptide of interest is CD19 or a fragment or mutant thereof.

In some embodiments, the fusion protein includes any of The amino acid sequence of one has an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical.

In some embodiments, the invention provides a nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of a fusion protein comprising (i) an antibody that binds a tumor antigen or an antigen-binding fragment thereof; and (ii) a polypeptide of interest. . In one aspect, the invention provides a fusion protein comprising (i) an antibody that binds a tumor antigen or an antigen-binding fragment thereof; (ii) a polypeptide of interest; and (iii) a half-life extending polypeptide. In some embodiments, the invention provides vectors comprising nucleic acids encoding fusion proteins described herein. In some embodiments, the invention provides host cells comprising nucleic acids encoding fusion proteins described herein. In some embodiments, the invention provides a method of producing a fusion protein described herein, comprising culturing a host cell comprising a nucleic acid encoding a fusion protein described herein. In some embodiments, the invention provides methods of treating an individual suffering from a tumor, comprising administering to the individual an effective amount of a fusion protein described herein.

In some embodiments, the invention provides an antibody or an antigen-binding fragment thereof, the antibody or an antigen-binding fragment thereof comprising an amino acid sequence having any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53 VHH or fragments thereof. In some embodiments, the invention provides nucleic acids encoding antibodies, or antigen-binding fragments thereof, comprising an amine having any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53 VHH or fragments thereof. In some embodiments, the invention provides a vector comprising a nucleic acid encoding an antibody or an antigen-binding fragment thereof having any one of SEQ ID NOs: 37, 39, 40, 42-44, and 53 The amino acid sequence of VHH or its fragment. In some embodiments, the invention provides a host cell comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof comprising any of SEQ ID NOs: 37, 39, 40, 42-44, and 53. An amino acid sequence of VHH or a fragment thereof. In some embodiments, the invention provides a method of producing an antibody or antigen-binding fragment thereof, the method comprising culturing a host cell comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising SEQ ID NO: VHH or a fragment thereof of the amino acid sequence of any one of 37, 39, 40, 42-44 and 53.

Other features, objects, and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments of the invention, are given by way of illustration only and not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

definition

In order to make the present invention easier to understand, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout this specification.

Administer : As used herein, the term "administer" refers to the administration of a composition to an individual or system. Administration to an individual animal (eg, human) may be by any appropriate route. For example, in some embodiments, administration can be transbronchial (including by bronchial instillation), buccal, enteral, intercutaneous, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, Intraperitoneal, intrathecal, intravenous, intraventricular, intrahepatic), transmucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, transtracheal (including by intratracheal instillation) Note), transdermal, transvaginal and transvitreous. In some embodiments, administration can be intratumoral or peritumoral. In some embodiments, administration may involve intermittent dosing. In some embodiments, administration may involve continuous administration (eg, infusion) over at least a selected period of time.

Acceptor cell therapy : As used herein, "acceptor cell therapy" or "ACT" involves the transfer of immune cells with anti-tumor activity into cancer patients. In some embodiments, ACT is a treatment method that involves using lymphocytes with anti-tumor activity, expanding these cells to large numbers ex vivo, and infusing them into a cancer-bearing host. In some embodiments, ACT is a treatment method involving the use of lymphocytes with anti-tumor activity, their infusion into a cancer-bearing host, and their ex vivo expansion.

Agent: As used herein, the term "agent" may refer to any chemical class of compounds or entities, including, for example, polypeptides, nucleic acids, carbohydrates, lipids, small molecules, metals, or combinations thereof. It will be clear from the context that in some embodiments the agent may be or comprise a cell or organism, or a fraction, extract or component thereof. In some embodiments, the agent is or includes a natural product, in that it is found in and/or obtained from nature. In some embodiments, the agent is or includes one or more artificial entities in that it is designed, engineered and/or produced by the action of human hands and/or does not exist in nature. In some embodiments, the agent can be used in isolated or pure form; in some embodiments, the agent can be used in crude form. In some embodiments, potential agents are provided as a collection or library, which can be screened, for example, to identify or characterize active agents therein. Some specific examples of agents that may be used in accordance with the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA/RNA hybrids, antisense oligonucleotides, ribozymes), peptides, Peptide mimetics, etc. In some embodiments, the agent is or contains a polymer. In some embodiments, the agent is not a polymer and/or contains essentially no polymer. In some embodiments, the agent contains at least one polymeric moiety. In some embodiments, the agent lacks or is essentially free of any polymeric moieties.

Amelioration : As used herein, "amelioration" means preventing, reducing and/or alleviating an individual's condition, or improving an individual's condition. Improvement includes, but does not require, complete recovery or complete prevention of a disease, condition or disorder.

Amino acid : As used herein, the term "amino acid" in its broadest sense refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, the amino acid has the general structure _H2N -C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a d-amino acid; in some embodiments, the amino acid is a l-amino acid. "Standard amino acid" refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than standard amino acids, whether prepared synthetically or obtained from natural sources. As used herein, "synthetic amino acid" includes chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxyl and/or amine terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups and/or substitution with other chemical groups, which Chemical groups can alter the circulating half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. Amino acids may contain one or more post-translational modifications, such as modification with one or more chemical entities ( e.g. , methyl, acetate, acetyl, phosphate, formyl moiety, isoprenoid , sulfate ester group, polyethylene glycol moiety, lipid moiety, carbohydrate moiety, biotin moiety, etc. ) combination. The terms "amino acid" and "amino acid residue" are used interchangeably and may refer to free amino acids and/or amino acid residues of peptides. It is obvious from the context in which the term is used that it refers to either a free amino acid or a residue of a peptide.

Antibody : The term "antibody" as used herein is intended to include typical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. Furthermore, the term "antibody" as used herein may, in appropriate embodiments (unless otherwise stated or apparent from context), refer to any construct or format known or developed in the art for use in alternative presentations of antibody structures. and functional characteristics. For example, in some embodiments, the form of antibodies used according to the invention is selected from, but not limited to, intact IgG, IgE and IgM, bispecific or multispecific antibodies (such as Zybodies® ^, etc.), bi- or multi-paratopic antibodies, Single-chain Fv, peptide-Fc fusion, Fab, camelid antibodies, masking antibodies (such as Probodies ^® ), small modular immunopharmaceuticals ("SMIPsTM"), single-chain or tandem diabatics (TandAb ^® ), VHH, Anticalins ^® , Nanobodies ^® , Microantibodies, BiTE ^® s, Ankyrin Repeat Proteins or DARPINs ^® , Avimers ^® , DART, TCR-like antibodies, Adnectins ^® , Affilins ^® , Trans-bodies ^® , Affibodies ^® , TrimerX ^® , MicroProteins, Fynomers ^® , Centyrins ^® and KALBITOR ^® . As is known in the art, naturally occurring intact IgG antibodies are approximately 150 kDa tetramers composed of two identical heavy chain polypeptides (each approximately 50 kDa) associated with each other in a structure commonly referred to as a "Y-shaped" structure. kDa) and two identical light chain polypeptides (each approximately 25 kDa). Each heavy chain consists of at least four domains (each about 110 amino acids in length): an amine-terminal variable (VH) domain (at the tip of the Y structure), followed by three constant domains: CH1, CH2 and carboxyl terminal CH3 (located at the base of the Y stem). A short region called a "switch" connects the variable and constant regions of the heavy chain. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in the intact antibody. Each light chain consists of two domains: an amine-terminal variable (VL) domain, followed by a carboxyl-terminal constant (CL) domain, separated from each other by another "switch." The complete antibody tetramer consists of two heavy chain-light chain dimers, in which the heavy chain and the light chain are connected to each other by a single disulfide bond; the other two disulfide bonds connect the heavy chain hinge region to each other, so that the two The polymers are connected to each other and form tetramers. Naturally occurring antibodies are also glycosylated, usually on the CH2 domain. Each domain in a natural antibody has a structure characterized by the "immunoglobulin fold," which consists of two beta sheets (e.g., 3, 4, or 5-stranded sheets) intersecting in compressed antiparallel beta barrels. formed by accumulation. Each variable domain contains three hypervariable loops called "complementarity determining regions" (CDR1, CDR2 and CDR3) and four "framework" regions that are somewhat invariant (FR1, FR2, FR3 and FR4 ). When a natural antibody folds, the FR region forms a beta sheet that provides the structural framework for these domains, and the CDR loop regions from the heavy and light chains come together in three dimensions so that they create the Y structure at the tip. Single hypervariable antigen binding site. The Fc region of naturally occurring antibodies binds elements of the complement system and also binds to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and/or other binding properties of the Fc region of an Fc receptor can be modulated via glycosylation or other modifications. In some embodiments, antibodies produced and/or used according to the present invention include glycosylated Fc domains, including Fc domains having modified or engineered such glycosylation. For purposes of this invention, in certain embodiments, any polypeptide or polypeptide complex that includes sufficient immunoglobulin domain sequences found in natural antibodies may be referred to and/or used as an "antibody," regardless of whether Polypeptides may be produced naturally (for example, by the reaction of an organism with an antigen), or may be produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, the antibodies are polyclonal; in some embodiments, the antibodies are monoclonal. In some embodiments, the antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are fully human, or humanized, primatized, chimeric, etc., as known in the art. In some embodiments, the antibody may lack covalent modifications (eg, attachment of glycans) that it would have if naturally occurring. In some embodiments, antibodies may contain covalent modifications (e.g., attachment of glycans, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.)). In some embodiments, protein scaffolds generated to bind the target antigen can be used as antigen-binding fragments. In some embodiments, protein binding molecules can be generated in silico based on the amino acid sequence and/or resolved structure of the protein of interest (e.g., Cao et al., Design of protein binding proteins from target structure alone. Nature, 2022; DOI : 10.1038/s41586-022-04654-9).

Antibody-Dependent Cytotoxicity : As used herein, the term "antibody-dependent cytotoxicity" or "ADCC" refers to the phenomenon in which target cells bound by an antibody are killed by immune effector cells. Without wishing to be bound by any particular theory, ADCC is generally understood to involve effector cells bearing Fc receptors (FcR) that recognize and subsequently kill antibody-coated target cells (e.g., cells expressing the antibody on their surface). cells that bind specific antigens). Effector cells that mediate ADCC may include immune cells, including but not limited to one or more of natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

Antibody fragment : As used herein, "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding region or variable region of the antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; triplexes; quadruplexes; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments, "Fv" fragments (composed of the variable regions of a heavy chain and a light chain), and recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are linked by a peptide linker ("scFv protein"). ”), recombinant single domain antibodies composed of antibody heavy chain variable regions (such as VHH), and hypervariable regions that mimic hypervariable regions (such as heavy chain variable regions (VH), light chain variable regions (VL) ), the smallest recognition unit consisting of amino acid residues in the hypervariable region, one or more CDR domains in VH and/or one or more CDR domains in VL). In many embodiments, the antibody fragment contains sufficient parent antibody sequence that it is a fragment that binds to the same antigen as the parent antibody; in some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and/or Competes with the parent antibody for binding to the antigen. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab') ₂ fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, heavy chain fragments, Variable regions and isolated complementarity determining region (CDR) regions. Antigen-binding fragments of antibodies can be produced by any means. For example, antigen-binding fragments of an antibody can be produced enzymatically or chemically by fragmentation of an intact antibody and/or they can be produced recombinantly from genes encoding partial antibody sequences. Alternatively or additionally, antigen-binding fragments of antibodies may be produced synthetically, in whole or in part. Antigen-binding fragments of antibodies optionally include single-chain antibody fragments. Alternatively or additionally, an antigen-binding fragment of an antibody may comprise multiple chains linked together, for example, by disulfide linkages. Antigen-binding fragments of antibodies optionally include multimolecular complexes. Functional antibody fragments typically contain at least about 50 amino acids and more typically at least about 200 amino acids.

Antigen: The term "antigen" as used herein refers to an agent that elicits an immune response; and/or binds to a T cell receptor (eg, when presented by an MHC molecule) or an antibody or antibody fragment. In some embodiments, the antigen elicits a humoral response (eg, including the production of antigen-specific antibodies); in some embodiments, the antigen elicits a cellular response (eg, involving T cells whose receptors specifically interact with the antigen). In some embodiments, the antigen binds the antibody and may or may not induce a specific physiological response in the organism. Generally, an antigen may be or include any chemical entity, such as a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments, other than a biopolymer (e.g., other than a nucleic acid or amino acid polymer)) wait. In some embodiments, the antigen is or comprises a polypeptide. In some embodiments, the antigen is or contains a glycan. Those skilled in the art will understand that, generally speaking, antigens may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, e.g., in an extract, such as cells containing the source of the antigen). extracts or other relatively crude preparations), or may be present on or within cells. In some embodiments, the antigen is a recombinant antigen.

About or Approximately: As used herein, the term "approximately" or "approximately" when applied to one or more values of interest refers to a value that is similar to the stated reference value. In certain embodiments, the term "about" or "approximately" means falling within 25%, 20%, 19%, 18%, 17%, 16%, 18%, 17%, 16%, Within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less Range of values, unless stated otherwise or otherwise obvious from context (except when the number exceeds 100% of possible values).

Binding : It is understood that, as used herein, the term "binding" generally refers to a non-covalent association between or among two or more entities. "Direct" bonding involves physical contact between entities or parts; indirect bonding involves physical interaction via physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., in covalent or When otherwise associated with a carrier entity and/or in a biological system or cell).

Cancer : The terms "cancer", "malignant disease", "neoplastic", "tumor" and "carcinoma" are used interchangeably herein to refer to a manifestation of relatively abnormal, uncontrolled and/or autonomous growth such that it exhibits Cells characterized by an abnormal growth phenotype characterized by significant loss of control over cell proliferation. Generally speaking, cells of interest for detection or treatment herein include precancerous (eg, benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. The teachings of this invention may be relevant to any and all cancers. To name just a few non-limiting examples, in some embodiments, the teachings of the present invention apply to one or more cancers, such as hematopoietic cancers, including leukemia, lymphoma (Hodgkin's tumor and non-Hodgkin's tumor). ), myeloma and myeloproliferative disorders; sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinomas of the mouth, throat, larynx and lungs, liver cancer, genitourinary system cancers such as prostate cancer, cervical cancer, bladder cancer Cancer, uterine cancer, endometrial cancer and renal cell cancer, bone cancer, pancreatic cancer, skin cancer, skin or intraocular melanoma, endocrine system cancer, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer and Cancers of the nervous system, benign lesions such as papillomas and the like.

Chimeric Antigen Receptor (CAR) As used herein, "chimeric antigen receptor" or "CAR" refers to a recombinant cell surface receptor engineered to express on immune effector cells and specifically target cells and/or or bind to antigen.

Representation : As used herein, "representation" of a nucleic acid sequence refers to one or more of the following events: (1) generation of an RNA template from a DNA sequence ( e.g. , by transcription); (2) processing of an RNA transcript ( e.g. , By splicing, editing, 5' cap formation and/or 3' end formation, etc.); (3) Translation of RNA into polypeptides or proteins; and/or (4) Post-translational modification of polypeptides or proteins.

Fusion Protein : As used herein, the term "fusion protein" generally refers to a polypeptide comprising at least two segments, each segment showing a high degree of amino acid identity to a peptide moiety that: (1) occurs in nature, and/or (2) Represents the functional domain of a polypeptide. In general, a polypeptide containing at least two such fragments is considered a fusion protein if the two fragments are part of the same peptide are interconnected in the polypeptide, and/or (3) have been interconnected by the action of human hands.

Nucleic acid : As used herein, "nucleic acid" in its broadest sense refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, the nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be apparent from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (eg, nucleotides and/or nucleosides); in some embodiments, "nucleic acid" refers to individual nucleic acid residues. oligonucleotide chain. In some embodiments, a "nucleic acid" is or includes RNA; in some embodiments, a "nucleic acid" is or includes DNA. In some embodiments, a nucleic acid is, contains, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, contains, or consists of one or more nucleic acid analogs. In some embodiments, nucleic acid analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, contains, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds in place of phosphodiester bonds in the backbone, being considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages instead of phosphodiester linkages. In some embodiments, the nucleic acid is, contains, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine glycosides, and deoxycytidine). In some embodiments, the nucleic acid is, contains, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine , 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodine Uridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8 -oxyadenosine, 8-oxyguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, embedded bases, and combinations thereof). In some embodiments, the nucleic acid includes one or more modified sugars (eg, 2'-fluoribose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to sugars in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product, such as RNA or protein. In some embodiments, the nucleic acid includes one or more introns. In some embodiments, the nucleic acid is synthesized by one or more of isolation from natural sources, enzymatic synthesis (in vivo or in vitro) by polymerization based on complementary templates, propagation in recombinant cells or systems, and chemical synthesis. to prepare. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 in length ,75,80,85,90,95,100,110,120,130,140,150,160,170,180,190,20,225,250,275,300,325,350,375,400,425 , 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, the nucleic acid is single-stranded; in some embodiments, the nucleic acid is double-stranded. In some embodiments, a nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

Pharmaceutically Acceptable : As used herein, the term "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for contact with human and animal tissue without undue toxicity, irritation, allergic reactions or other problems or complications and are commensurate with a reasonable benefit/risk ratio.

Polypeptide : As used herein, a "polypeptide" is generally a string of at least two amino acids linked to each other by peptide bonds. In some embodiments, a polypeptide may include at least 3-5 amino acids, each amino acid being linked to other amino acids by at least one peptide bond. In some embodiments, the polypeptide can be longer than 5 amino acids, with each amino acid linked to other amino acids by at least one peptide bond. Those skilled in the art will understand that polypeptides sometimes include "non-natural" amino acids or other entities that can still be integrated into the polypeptide chain as appropriate.

Protein: As used herein, the term "protein" refers to a polypeptide (ie, a string of at least two amino acids linked to each other by peptide bonds). Proteins may include moieties other than amino acids (eg, they may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those skilled in the art will recognize that a "protein" may be an entire polypeptide chain produced by a cell (with or without a signal sequence), or may be a portion thereof. Those skilled in the art will recognize that a protein may sometimes include more than one polypeptide chain linked, for example, by one or more disulfide bonds or otherwise associated. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, sialylation, acetylation, amidation, methylation, phosphorylation, and the like. In some embodiments, proteins can include natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

Individual : "Individual" means a mammal (such as a human). In some embodiments, the individual suffers from a related disease, condition or disorder. In some embodiments, the individual is susceptible to a disease, condition or disorder. In some embodiments, an individual exhibits one or more symptoms or characteristics of a disease, condition, or disorder. In some embodiments, the individual does not exhibit any symptoms or characteristics of the disease, condition, or disorder. In some embodiments, an individual is a person who has one or more characteristics that characterize susceptibility or risk for a disease, condition, or disorder. In some embodiments, the individual is a patient. In some embodiments, the subject is a subject that is administered and/or has been administered a diagnosis and/or therapy.

Affected : An individual "suffering from" a disease, condition or disorder ( such as cancer) has been diagnosed with and/or exhibits one or more symptoms of the disease, condition or condition.

Therapeutically Effective Amount: As used herein, the term "therapeutically effective amount" means an amount sufficient to treat the disease, disorder, and/or disorder when administered to a population suffering from or susceptible to the disease, disorder, and/or disorder, according to a therapeutic dosage regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays the onset of one or more symptoms of a disease, disorder, and/or disorder. Those skilled in the art will understand that the term "therapeutically effective amount" is not actually required to achieve successful treatment in a particular individual. Rather, a therapeutically effective amount may be an amount that provides a specific desired pharmacological response in a large number of individuals when administered to a patient in need of such treatment. For example, in some embodiments, a "therapeutically effective amount" means one that, when administered to an individual in need thereof in the context of a therapy of the present invention, blocks, stabilizes, attenuates, or reverses the cancer-supportive processes occurring in that individual, or An amount that will enhance or increase the cancer suppressive process in the individual. In the context of cancer treatment, a "therapeutically effective amount" refers to an amount that, when administered to an individual diagnosed with cancer, will prevent, stabilize, inhibit, or reduce the further development of cancer in the individual. Particularly preferred "therapeutically effective amounts" of the compositions described herein reverse (in treatment) the development of a malignant tumor, such as pancreatic cancer, or assist in achieving or prolonging remission of the malignant disease. The therapeutically effective amount administered to an individual to treat cancer in that individual may be the same as or different from the therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the treatments described herein should not be construed, limited or otherwise limited to the "cure" of cancer; rather, the treatments involve the use of the compositions described to "treat" Cancer, that is, a desired or beneficial change in the health of an individual suffering from cancer. Such benefits have been recognized by healthcare providers skilled in the field of oncology and include, but are not limited to, stabilization of patient condition, reduction in tumor size (tumor regression), improvement in vital functions (e.g., improvement in the function of cancerous tissues or organs). ), reduction or inhibition of further metastasis, reduction of opportunistic infections, increase in viability, reduction in pain, improvement in motor function, improvement in cognitive function, improvement in sense of energy (reduction in vitality, discomfort), and sense of well-being Improvement, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. Additionally, regression of a particular tumor in an individual (e.g., as a result of treatment described herein) can also be assessed by collecting a sample of cancer cells from a tumor site, such as pancreatic cancer (e.g., during treatment) and testing Metabolic and signaling marker levels of cancer cells are used to monitor the status of cancer cells to verify the regression of cancer cells to a less malignant phenotype at the molecular level. For example, tumor regression induced by use of the methods of the invention will be indicated by the finding of: a decrease in one or more pro-angiogenic markers, an increase in an anti-angiogenic marker, in an individual diagnosed with cancer Normalization (i.e., change to the state found in normal individuals without cancer) of metabolic pathways, intercellular signaling pathways, or intracellular signaling pathways that exhibit abnormal activity. It will be understood by those skilled in the art that in some embodiments, a therapeutically effective amount may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered in multiple doses, eg, as part of a dosing regimen.

Treatment : As used herein, the term "treatment" (also "treat" or "treating") refers to any administration that partially or completely alleviates, ameliorates, alleviates, inhibits a specific disease, condition, and A substance that delays, delays the onset of, reduces the severity of, and/or reduces the incidence of, one or more symptoms, characteristics and/or causes of a disease (such as cancer). Such treatment may be directed to individuals who do not exhibit signs of the relevant disease, disorder, and/or disorder, and/or to individuals who only exhibit early signs of the disease, disorder, and/or disorder. Alternatively or additionally, such treatment may be directed to individuals exhibiting one or more established signs of the relevant disease, condition and/or disorder. In some embodiments, treatment may be directed to an individual who has been diagnosed with a relevant disease, disorder, and/or disorder. In some embodiments, treatment may be for individuals known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, condition, and/or disorder.

Vector : As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid in association with it. In some embodiments, the vector is capable of extrachromosomal replication and/or expression of the nucleic acid to which it is linked in a host cell, such as a eukaryotic and/or prokaryotic cell. A vector capable of directing the expression of an operably linked gene is referred to herein as an "expression vector." A detailed description of specific examples

With the introduction of autologous T cells transduced with chimeric antigen receptors (CARs) targeting the B-cell malignancy antigen CD19, the future of cancer cell therapy has become clear. Anti-CD19 CAR-T cells (CAR-19s) induce high response rates and durable remissions in patients with relapsed and refractory acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) (Ruella et al., Curr Hematol Malig Rep. 2016;11:368–84). Long-term follow-up studies have demonstrated the potential for cure, with some patients approaching 10 years cancer-free (Chong et al., New England Journal of Medicine. Massachusetts Medical Society; 2021; 384:673–4; Neelapu et al., Blood. 2021; 138:93 ; Cappell et al., J Clin Oncol. 2020; 38:3805–15; Schuster et al., Biology of Blood and Marrow Transplantation. 2019; 25:S20–1; Grupp et al., Biology of Blood and Marrow Transplantation. Elsevier; 2019 ; 25:S126–7).

However, the cost of CAR-19 therapy remains high, and the necessary infrastructure limits the environments in which CAR can be produced and administered (Lin et al., J Clin Oncol. 2019; 37:2105–19). Preparative hemopheresis, consolidation, lymphocyte depletion, and toxicity can limit the use of CAR-T therapy in frail patients. Side effects of CAR-19 therapy can be significant, including grade 3+ cytokine release syndrome (CRS) and immune effector cell-associated neurotoxic syndrome (ICANS), which can lead to prolonged hospitalization and additional cost of care (Brudno et al., Blood Rev. 2019;34:45–55). More than half of patients who respond subsequently relapse, often within months of treatment (Maude et al., New England Journal of Medicine. Massachusetts Medical Society; 2018; 378:439–48; Neelapu et al., New England Journal of Medicine. Massachusetts Medical Society; 2017;377:2531–44). Many patients relapse due to loss or downregulation of the CD19 protein on leukemia or lymphoma cells (Shah et al., Nat Rev Clin Oncol. 2019; 16:372–85).

Antigen loss and recurrence is a phenomenon driven by natural selection, that is, CAR-19 T cells can exert strong selective pressure on the malignant B cell population, thereby driving the selection of pure lines with loss or reduction of the target protein CD19. Antigen loss can occur via mutational events that prevent expression of the CD19 extracellular domain (ECD) or regions within the ECD and/or through transcriptional downregulation of expression (Plaks et al., Blood. 2021; 138:1081–5; Majzner et al., Cancer Discov. American Association for Cancer Research; 2018; 8:1219–26). Such escape mechanisms are common in cancer treatment modalities (Donk et al., Blood Cancer Discov. American Association for Cancer Research Journals; 2021; 2:302–18; Ruella et al., Comput Struct Biotechnol J. 2016; 14: 357–62). Other mechanisms of evasion of CAR-19 therapy have been described and include lineage switch to myeloid leukemia and trogocytosis, a process in which CD19 is physically stripped from lymphoma cells by CAR (Perna, Translational Cancer Research [Internet] . AME Publishing Company; 2016 [cited January 12, 2022]; 5 (available at tcr.amegroups.com/article/view/9016); Miao et al., Frontiers in Immunology. 2021; 12:1862).

The fusion proteins described herein provide solutions to one or more of these problems. In some embodiments, the fusion proteins described herein can be expressed by cells transduced with lentiviral, retroviral or other gene therapy vectors, and/or can be expressed by in vitro mammalian cell cultures and subsequently subjected to Purified to produce injectable biologics. In some embodiments, the fusion protein of the invention includes three functional domains: modified CD19 ECD, anti-CD20 binding domain, and anti-albumin binding domain. In some such embodiments, the fusion proteins of the invention bind CD20 and display CD19 ECD. As described herein, such fusion proteins can (i) increase CD19 antigen density on target tumor cells regardless of their level of CD19 expression, and (ii) effectively trigger CD19-negative tumors in the presence of CAR-19 T cells in vitro cell death, (iii) prevent antigen loss relapse to evade CAR-19 therapy, (iv) prevent CD19-negative tumor expansion, e.g. at relatively low doses, and/or (v) have a significant impact on survival. As described herein, the fusion proteins of the invention can be expressed by transfected mammalian cells, can be efficiently purified, and exhibit favorable biophysical properties, indicating that such fusion proteins may be suitable for scale-up and production. fusion protein

In some embodiments, the invention provides fusion proteins comprising or consisting of: (i) a first antigen-binding polypeptide that binds a first tumor antigen and (ii) a polypeptide of interest. In some embodiments, the invention provides a fusion protein comprising or consisting of: (i) a first antigen-binding polypeptide that binds a first tumor antigen, (ii) a second antigen-binding polypeptide that binds a second tumor antigen. , and (iii) target polypeptide. In some embodiments, the fusion proteins described herein comprise or consist of: and SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, Any one of 28, 30, 32, 34, 36, 50 and 52 is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, but the amino acid sequence of the disclosed half-life extending polypeptide is missing in each sequence. In some embodiments, the fusion proteins described herein comprise or consist of: at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, but lacking the disclosed half-life extending polypeptide in each sequence and Amino acid sequences lacking the disclosed C-terminal hexahistidine tag. In some embodiments, the fusion proteins described herein comprise or consist of at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of SEQ ID NO. 55 , 97%, 98%, 99% or 100% identical amino acid sequences.

In some embodiments, the invention provides fusion proteins comprising or consisting of: (i) an antigen-binding polypeptide that binds a tumor antigen, (ii) a polypeptide of interest, and (iii) a half-life extending polypeptide. In some embodiments, the invention provides fusion proteins comprising or consisting of: (i) a first antigen-binding polypeptide that binds a first tumor antigen, (ii) a second antigen-binding polypeptide that binds a second tumor antigen, (iii) a target polypeptide, and (iv) a half-life extending polypeptide. In some embodiments, the fusion proteins described herein comprise or consist of: and SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, Any one of 28, 30, 32, 34, 36, 50 and 52 is at least approximately 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence. As shown in the Sequence Listing provided herein, each of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, and 50 includes (amino to carboxyl terminus): signal sequence; anti- CD20 VHH; first linker (GSGGGGSGGGGS); target polypeptide; second linker (SRGGGGGSGGGGSGGGGS); half-life extending polypeptide; and hexahistidine tag. In some embodiments, the fusion proteins described herein comprise or consist of: at least about Amino acids that are 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical and lack the C-terminal hexahistidine tag sequence. As shown in the sequence listing provided herein, each of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, and 52 includes (amine to carboxyl terminus): anti-CD20 VHH; The first linker (GSGGGGSGGGGS); the target polypeptide; the second linker (SRGGGGGSGGGGSGGGGS); and the half-life extending polypeptide.

In addition to those described herein, those skilled in the art will recognize many amino acid sequences suitable for use as linkers. In some embodiments, the first and/or second linker comprise a series of naturally occurring amino acids. In some embodiments, the linker is derived from a naturally occurring multi-domain protein. In some embodiments, the connector is flexible. In some embodiments, flexible linkers are designed using computational tools familiar to those skilled in the art, such as JPred (Dorzdetskiy et al., Nucl Acids Res. 2015; 43: W389-94). In some embodiments, the linker adopts a preferred configuration. In some embodiments, the linker includes a GS linker, wherein the linker includes an amino acid sequence of the formula [G _{x Sy} ] _z , where x, y, and z are positive integers. In some embodiments, the linker comprises a polyglycine linker, wherein the linker comprises the amino acid sequence _Gx , where x is a positive integer. In some embodiments, the linker comprises a glycine- and serine-rich linker, wherein glycine and serine together comprise 50% or more of the linker's amino acid sequence. Many examples and applications of specific linker sequences in fusion proteins and linker design methods are described in Chen et al., Adv Drug Deliv Rev. 2013; 65: 1357-69; and Liu et al., Bioinformatics. 2015; 31: 3700- 2, their respective contents are incorporated herein by reference. Antigen binding peptide

In some embodiments, fusion proteins of the invention include antigen-binding polypeptides that bind a tumor antigen, such as a tumor antigen described herein. In some embodiments, the antigen-binding polypeptide binds CD20. In some embodiments, the antigen-binding polypeptide is an anti-CD20 antibody or CD20-binding fragment thereof. In some embodiments, the antigen-binding polypeptide is or comprises an anti-CD20 scFv, Fv, or other multi-domain binding fragment. In some embodiments, the antigen-binding polypeptide is or comprises an anti-CD20 single domain antibody, such as an anti-CD20 single domain antibody described herein.

In some embodiments, fusion proteins of the invention include a first antigen-binding polypeptide that binds CD20, and a second tumor antigen described herein (e.g., CD79b, HER-2/neu, c-met, EGFR, Ga733, EpCAM , CD21, ROR1, CLL-1/CLEC12A, HLA-DR10, CD1, CD5, CD21, CD25, CD27, CD30, CD38, CD78, CD80, CD86, CD138, CD319, surface Ig, PD-1, PD-L1, PD-L2, TGFbR2 or BCMA) second antigen-binding polypeptide. In some embodiments, the first antigen-binding polypeptide is an anti-CD20 antibody or CD20-binding fragment thereof. In some embodiments, the first antigen-binding polypeptide is or comprises an anti-CD20 scFv, Fv, or other multi-domain binding fragment. In some embodiments, the first antigen-binding polypeptide is or comprises an anti-CD20 single domain antibody, such as an anti-CD20 single domain antibody described herein. single domain antibody

Single-domain antibodies are antibodies whose complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional 4 chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. The single domain antibody can be any known in the art, or any future single domain antibody. Single domain antibodies can be derived from any species, including but not limited to mouse, human, camel, llama, goat, rabbit, cow. According to one aspect of the invention, the single domain antibody system used herein is referred to as a single domain antibody that does not contain a light chain heavy chain antibody. Such single domain antibodies are disclosed, for example, in WO 94/04678. Variable domains derived from heavy chain antibodies that naturally do not contain light chains (eg, single domain antibodies) are referred to herein as "VHHs" or "nanobodies." Such VHHs may be derived from antibodies produced in Camelidae species, such as camels, dromedaries, llamas, vicuñas, alpacas, and guanacos. Other species outside the family Camelidae may produce heavy chain antibodies that naturally lack light chains; such VHHs are within the scope of the invention.

Amino acid residues of VHH domains from camelids are numbered according to the general numbering of VH domains given in: Kabat et al., "Sequence of proteins of immunological interest", US Public Health Services, NIH (Bethesda, MD), Publication No. 91–3242 (1991); see also Riechmann et al., J. Immunol. Methods 231:25-38 (1999). According to this numbering, FR1 contains the amino acid residues at positions 1-30, CDR1 contains the amino acid residues at positions 31-35, FR2 contains the amino acid residues at positions 36-49, and CDR2 contains the amino acid residues at positions 50-65. As for amino acid residues, FR3 includes amino acid residues at positions 66-94, CDR3 includes amino acid residues at positions 95-102, and FR4 includes amino acid residues at positions 103-113.

However, it should be noted (as is well known in the art for VH domains and VHH domains) that the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (That is, one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows). This means that in general, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.

Alternative methods for numbering the amino acid residues of VH domains are known in the art and can be applied to VHH domains in a similar manner. Unless otherwise stated, in the present invention, patent claims and drawings, CDRs are numbered according to IMGT (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38:D301-D307 (2010 ) to define. Anti -CD20 VHH

In some embodiments, fusion proteins of the invention include a CD20-binding polypeptide that is or includes an anti-CD20 VHH. In some embodiments, the anti-CD20 VHH comprises or consists of the amino acid sequence of any of SEQ ID NOs: 37-44 and 53, or a CD20-binding fragment thereof.

In some embodiments, the anti-CD20 VHH comprises or consists of at least 85%, 90%, 91% the amino acid sequence of any one of SEQ ID NOs: 37-44 and 53, or a CD20-binding portion thereof , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.

In some embodiments, an anti-CD20 VHH comprises or consists of at least one CDR (eg, CDR1, CDR2, and/or CDR3) described in any of SEQ ID NOs: 37-44 and 53. In some embodiments, the anti-CD20 VHH includes CDR1, CDR2, and CDR3 described in SEQ ID NO:37. In some embodiments, the anti-CD20 VHH includes CDR1, CDR2, and CDR3 described in SEQ ID NO:38. In some embodiments, the anti-CD20 VHH includes CDR1, CDR2, and CDR3 described in SEQ ID NO:39. In some embodiments, the anti-CD20 VHH includes CDR1, CDR2, and CDR3 described in SEQ ID NO:40. In some embodiments, the anti-CD20 VHH includes CDR1, CDR2, and CDR3 described in SEQ ID NO:41. In some embodiments, the anti-CD20 VHH includes CDR1, CDR2, and CDR3 described in SEQ ID NO:42. In some embodiments, the anti-CD20 VHH includes CDR1, CDR2, and CDR3 described in SEQ ID NO:43. In some embodiments, the anti-CD20 VHH includes CDR1, CDR2, and CDR3 described in SEQ ID NO:44. In some embodiments, the anti-CD20 VHH includes CDR1, CDR2, and CDR3 described in SEQ ID NO:53.

In some embodiments, an anti-CD20 VHH comprises or consists of at least 85%, 90% similarity to the CDRs (e.g., CDR1, CDR2, and/or CDR3) described in any of SEQ ID NOs: 37-44 and 53. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% consistent with at least one CDR. In some embodiments, the anti-CD20 VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the CDR1 described in SEQ ID NO:37 , CDR1 that is 99% or 100% identical; at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the CDR2 described in SEQ ID NO:37 , 99% or 100% identical CDR2; and at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the CDR3 described in SEQ ID NO:37 %, 99% or 100% consistent CDR3. In some embodiments, the anti-CD20 VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the CDR1 described in SEQ ID NO:38 , CDR1 that is 99% or 100% identical; at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the CDR2 described in SEQ ID NO:38 , 99% or 100% identical CDR2; and at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the CDR3 described in SEQ ID NO:38 %, 99% or 100% consistent CDR3. In some embodiments, the anti-CD20 VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the CDR1 described in SEQ ID NO:39 , 99% or 100% identical CDR1; at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to the CDR2 described in SEQ ID NO:39 , 99% or 100% identical CDR2; and at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the CDR3 described in SEQ ID NO:39 %, 99% or 100% consistent CDR3. In some embodiments, the anti-CD20 VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the CDR1 described in SEQ ID NO:40 , CDR1 that is 99% or 100% identical; at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the CDR2 described in SEQ ID NO:40 , 99% or 100% identical CDR2; and at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the CDR3 described in SEQ ID NO:40 %, 99% or 100% consistent CDR3. In some embodiments, the anti-CD20 VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the CDR1 described in SEQ ID NO:41 , CDR1 that is 99% or 100% identical; at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the CDR2 described in SEQ ID NO:41 , 99% or 100% identical CDR2; and at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the CDR3 described in SEQ ID NO:41 %, 99% or 100% consistent CDR3. In some embodiments, the anti-CD20 VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the CDR1 described in SEQ ID NO:42 , 99% or 100% identical CDR1; at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to the CDR2 described in SEQ ID NO:42 , 99% or 100% identical CDR2; and at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the CDR3 described in SEQ ID NO:42 %, 99% or 100% consistent CDR3. In some embodiments, the anti-CD20 VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the CDR1 described in SEQ ID NO:43 , CDR1 that is 99% or 100% identical; at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the CDR2 described in SEQ ID NO:43 , 99% or 100% identical CDR2; and at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the CDR3 described in SEQ ID NO:43 %, 99% or 100% consistent CDR3. In some embodiments, the anti-CD20 VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the CDR1 described in SEQ ID NO:44 , CDR1 that is 99% or 100% identical; at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the CDR2 described in SEQ ID NO:44 , 99% or 100% identical CDR2; and at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the CDR3 described in SEQ ID NO:44 %, 99% or 100% consistent CDR3. In some embodiments, the anti-CD20 VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the CDR1 described in SEQ ID NO:53 , CDR1 that is 99% or 100% identical; at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the CDR2 described in SEQ ID NO:53 , 99% or 100% identical CDR2; and at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the CDR3 described in SEQ ID NO:53 %, 99% or 100% consistent CDR3.

Antibodies or fragments can be produced by any method known in the art for the synthesis of antibodies (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et al. , 1995, J. Immunol. Methods 182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can be produced, for example, using methods described in Morrison, 1985, Science 229:1202, and humanized antibodies can be produced, for example, by methods described in US Pat. No. 6,180,370. Target peptide

In some embodiments, fusion proteins of the invention include a polypeptide of interest. In some embodiments, the polypeptide of interest is an antigen target for a cell therapy, such as a CAR-T cell, an antibody, or an antibody drug conjugate as described in WO2017/075537, WO2017/075533, WO2018156802, and WO2018156791.

In some embodiments, the polypeptide of interest includes or consists of all or part of a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens , such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER- 2/neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus Antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other tumor antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, erbB, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19 -9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG , BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp- 175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP , MUC16, IL13Rα2, FRα, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, Nectin-4, AGS-16, guanylate ring enzyme C, MUC-1, CFC1B, integrin α3 chain (belonging to laminin receptor chain a3b1) and TPS.

In some embodiments, the target polypeptide includes or consists of all or part of a tumor antigen selected from: CD19, CD20, CD22, CD30, CD72, CD180, CD171 (L1CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1/CLEC12A, ROR1, glypican 3 (GPC3), mesothelin, CD33/IL3Ra, c-Met, PSCA, PSMA, glycolipid F77 , EGFRvIII, GD-2, MY-ESO-1 and MAGE A3.

In some embodiments, the polypeptide of interest includes or consists of all or part of the following B cell specific markers: such as CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, CD180, ROR1, BCMA, HLA-DR10, CD1, CD5, CD21, CD25, CD27, CD30, CD38, CD78, CD80, CD86, CD138, CD319, surface Ig, PD-1, PD-L1, PD-L2, TGFbR2, CD79a and CD79b (see e.g. LeBien et al. , Blood 112:1570-1580 (2008).

CD19 is a 95 kDa type I transmembrane glycoprotein used as a biomarker of B cell development (Wang et al., Exp. Hematol. Oncol. 1:36 (2012)). The expression of CD19 in lymphoma and leukemia makes it an effective therapeutic target, especially for chimeric antigen receptor (CAR) T cell therapy (Maude et al., Blood 125:4017–4024 (2015)). Based on the uniquely potent performance of CD19 in CAR-T cell therapy, treatments have been described that involve the use of antibody-CD19 fusions or CD19 variants engineered to directly bind tumor biomarkers to "transform ^" tumors. ” are CD19 ⁺ tumors (see, for example, WO2017/075537 and WO2017/075533).

The extracellular region of CD19 is hypothesized to contain two C2-like immunoglobulin domains (see, e.g., Wang et al., Exp. Hematol. Oncol. 1:36 (2012); Tedder et al., Nat. Rev. Rheumatol. 5:572– 577 (2009)). This is supported by homology models (Söding et al., Nucleic Acids Res. 33:244–248 (2005)). However, a later published structure showed that CD19 does not include a C2-like immunoglobulin domain (Teplyakov et al., Proteins 86:495-500 (2018)). The amino acid sequence of wild-type human CD19 is provided herein as SEQ ID NO:47.

In some embodiments, the target polypeptide includes or consists of all or part of the amino acid sequence of SEQ ID NO: 47. In some embodiments, the target polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 47 or consists of its composition. In some embodiments, the target polypeptide comprises or consists of about 200, 210, 220, 230, 240, 250, 260, 270 or 280 consecutive amino acids of SEQ ID NO: 47. In some embodiments, the polypeptide of interest comprises or consists of all or part of the CD19 extracellular domain (ECD). For example, in some embodiments, the polypeptide of interest comprises a fragment of SEQ ID NO: 47, for example, a fragment including or consisting of about amino acid 20 to about amino acid 278 of SEQ ID NO: 47. In some embodiments, the target polypeptide includes or consists of all or part of the amino acid of SEQ ID NO: 48. In some embodiments, the target polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 48 or consists of its composition. In some embodiments, the polypeptide of interest comprises or consists of all or part of a CD19 variant or fragment thereof. In some embodiments, the target polypeptide includes or consists of all or part of the amino acid of SEQ ID NO: 56. In some embodiments, the target polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 48 or consists of its composition. In some embodiments, the polypeptide of interest comprises or consists of all or part of a CD19 variant or fragment thereof. In some embodiments, a CD19 variant is or includes a full-length CD19 polypeptide (eg, SEQ ID NO:47) or a portion thereof that includes one or more amino acid substitutions described herein. In some embodiments, a CD19 variant is or includes a CD19 ECD (eg, SEQ ID NO:48 or SEQ ID NO:56) or a portion thereof that includes one or more amino acid substitutions. In some embodiments, the CD19 variant is or includes the CD19 ECD, or a portion thereof, described in WO2019/118918, the entire contents of which are incorporated herein by reference. In some embodiments, the CD19 variant comprises or consists of the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CD19 variant comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 46, or consists of. half-life extension

In some embodiments, fusion proteins of the invention include an agent that increases the half-life of the fusion protein, e.g., relative to a fusion protein that does not contain such agent. In some embodiments, such agents are polypeptides, referred to herein as "half-life extending polypeptides."

In some embodiments, the half-life extending polypeptide is a transferrin polypeptide or a portion thereof. Transferrin is recycled by binding to the transferrin receptor (see, eg, Widera et al., Adv. Drug Deliv. Rev. 55:1439-66 (2003)). In some embodiments, the half-life extending polypeptide is albumin (eg, bovine serum albumin (BSA), human serum albumin (HSA), or mouse serum albumin (MSA)) or a fragment thereof. In some embodiments, the half-life extending polypeptide is a polypeptide that binds serum protein. In some embodiments, the half-life extending polypeptide is a serum albumin binding agent (eg, a BSA, HSA, or MSA binding agent).

In some embodiments, the serum albumin binding agent is an albumin binding peptide. Albumin-binding peptides are described in WO200145746, WO2002076489, WO2008068280, WO2009127691, WO2011095545, and US Patent Publications Nos. 20040001827, 20080187517, and 20130316952. Those skilled in the art are familiar with methods of directly linking proteins and antibodies to albumin or albumin domains, for example in Patterson et al., Bioconjugate Chem. 2016, 27, 10, 2271–2275; Bern et al., Sci. Trans. Med., October 14, 2020 • Volume 12, Issue 565.

In some embodiments, the half-life extending polypeptide is or includes a hyaluronic acid binding domain. Hyaluronic acid (HA), also known as hyaluronic acid, is a glycosaminoglycan that exists in connective tissue and other tissues and is abundant in synovial fluid, skin and vitreous body. HA binds to a large number of naturally occurring hyaluronic acid binding proteins (HABP). Some HABPs contain an HA-binding domain, called a linker module, through which they bind to HA (Kohda, C.J. et al., Cell 86 (1996) 767-775.). Some HAPBs contain a linear 9–11 residue HA binding motif, which contains multiple basic amino acids called the B-X7-B motif (Yang B. et al. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. EMBO J., 13: 286-296, 1994).

In some embodiments, the half-life extending polypeptide is a PAS polypeptide. As used herein, a "PAS polypeptide" is a polypeptide characterized in that the sum of proline, alanine, and serine residues constitutes more than about 80%, or about 85%, of the total amino acid sequence of the half-life extending polypeptide. , or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or 100%. In general, PAS polypeptides are characterized by adopting a random coil conformation under physiological conditions, as described in U.S. Patent Publication No. 20100292130. PAS polypeptides useful as half-life extending polypeptides in fusion proteins of the invention are further described in WO 2008/155134, US Patent Publication No. 20100292130, US Patent No. 8,563,521, and/or US Patent No. 9,260,494.

In some embodiments, the half-life extending polypeptide consists solely of proline and alanine, or consists primarily of proline and alanine but may have up to 1%, 2%, 3%, 5%, or 10% of other amines acid residues. Where other amino acids are present, they may all be the same, or a plurality of different amino acids may be present. Examples of polypeptides composed primarily or entirely of proline and alanine that adopt a random coil conformation under physiological conditions (referred to as proline/alanine random coil polypeptides) are described in U.S. Patent Publication No. 20130072420 , U.S. Patent No. 9,221,882 and/or U.S. Patent No. 10,081,657.

In some embodiments, the half-life extending polypeptide is characterized in that the sum of glycine, alanine, serine, threonine, glutamine, and proline residues accounts for polypeptide of the total amino acid sequence of the half-life extending polypeptide. At about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or 100%, and the half-life extending polypeptide includes this At least 4 of 6 different amino acids. Such polypeptides may be said to consist essentially of amino acids selected from G, A, S, T, E, and P (see, eg, U.S. Patent Publication Nos. 20030228309; 9,926,351; 9,976,166; and 10,961,287).

In some embodiments, the half-life extending polypeptide is an antibody or fragment thereof. In some embodiments, the half-life extending polypeptide is an anti-albumin antibody or albumin-binding fragment thereof. In some embodiments, the half-life extending polypeptide is or comprises an anti-albumin single domain antibody. In some embodiments, the half-life extending polypeptide is or comprises an anti-albumin VHH. In some embodiments, the anti-albumin VHH comprises or consists of the amino acid sequence disclosed or described in US Publication No. 20070269422, the entire contents of which is incorporated herein by reference. In some embodiments, the anti-albumin VHH comprises or consists of the amino acid sequence of SEQ ID NO: 45 or the albumin-binding portion thereof. In some embodiments, the anti-albumin VHH comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of SEQ ID NO: 45 or an albumin-binding portion thereof , or consisting of an amino acid sequence that is 98%, 99% or 100% identical. tumor

The present invention provides techniques that can be used to treat any tumor. In some embodiments, the tumor is or includes a hematological malignancy, including but not limited to acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, AIDS-associated lymphoma, cholera Chickkin's lymphoma, non-Hodgkin's lymphoma, Langerhans cell histiocytosis, multiple myeloma, or myeloproliferative neoplasia.

In some embodiments, the tumor is or includes a solid tumor, including but not limited to breast cancer, squamous cell carcinoma, colorectal cancer, head and neck cancer, ovarian cancer, lung cancer, mesothelioma, genitourinary cancer, rectal cancer, gastric cancer, or esophageal cancer cancer.

In some specific embodiments, the tumor is or includes an advanced tumor and/or a refractory tumor. In some embodiments, when certain pathologies are observed in tumors (e.g., in tissue samples obtained from tumors, such as biopsy samples) and/or when cancer patients with such tumors are not generally considered to be When candidates for chemotherapy, tumors are characterized as advanced. In some embodiments, pathologies that characterize a tumor as advanced may include tumor size, altered expression of genetic markers, invasion of adjacent organs and/or lymph nodes by tumor cells. In some embodiments, when a patient with such a tumor is resistant to one or more known treatment modalities (e.g., one or more known chemotherapy regimens) and/or when a particular patient is resistant to one or more such known treatment modalities, Tumors are characterized as refractory when they exhibit resistance (eg, lack of responsiveness) to known treatment modalities. cell therapy

In some embodiments, the fusion proteins described herein can be administered to an individual as a cell therapeutic agent. For example, a nucleotide sequence encoding a fusion protein described herein can be introduced into a cell and administered to an individual as a cell therapeutic. In some embodiments, cell therapeutic agents can be produced from immune cells, such as cells that are or can be used in receptive cell therapy. In some embodiments, the cell therapeutic agent is produced from a cell type selected from the group consisting of: TIL, T cells, CD8 ⁺ cells, CD4 ⁺ cells, NK cells, gamma-delta T cells, regulatory T cells, iNKT cells , monocytes, macrophages, IPSC-derived cells or peripheral blood monocytes. As used herein, "tumor-infiltrating lymphocytes" or TILs refer to white blood cells that have left the bloodstream and migrated into tumors. Lymphocytes can be divided into three groups, including B cells, T cells and natural killer cells. As used herein, "T cells" refers to CD3 ⁺ cells, including CD4 ⁺ helper cells, CD8 ⁺ cytotoxic T cells, and delta-gamma T cells.

In certain embodiments, cell therapeutics are produced by genetically modifying (eg, transforming) cells, such as immune cells, with nucleic acids encoding fusion proteins described herein. In some embodiments, such nucleic acids are included in recombinant expression vectors. Recombinant expression vectors may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single- or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural or altered Nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring inter-nucleotide linkages, or both types of linkages.

The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors useful in the context of the present invention include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present invention include pcDNA, pEUK-Cl, pMAM and pMAMneo (Clontech).

In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, but are not limited to, retroviral vectors, alphaviruses, vaccine viruses, adenoviruses, adeno-associated viruses, herpesviruses, and fowlpox virus vectors, and preferably have the ability to transform immune cells (e.g., T cells) innate or Engineering capabilities.

In ex vivo applications such as cell therapy, gamma retroviral vectors derived from murine leukemia virus (MLV) were first developed and are still in use. Human immunodeficiency virus (HIV)-based lentiviral vectors are widely used. General strategies for designing lentiviral vectors are based on deletion and alteration of native viral sequences to prevent the generation of replication-competent viruses. Therefore, the lentiviral components are divided into three or four different plastid constructs in order to prevent the possibility of complete recombination into fully replication-competent lentivirus (RCL). The viral vector genome contains at least the transgene expression cassette, long terminal repeats (LTR) and packaging signals. In most cases, three additional plasmids provide factors required for virus production and packaging (eg, gag, pol, env). The promoter-enhancer region from the 3' LTR was also deleted, preventing transcription from this region and subsequent viral replication (termed a self-inactivating vector; SIN). The basic steps in ex vivo cell transformation or transduction include cell isolation and culture of the desired cell type for selection, expansion and differentiation before or after transducing the cells with viral vectors. In the case of hematopoietic cells, most of these steps are performed in a closed system using disposable blood collection and processing bags. For CAR T cell therapy, blood cells are collected from the patient, the desired T cell population is selected and grown to the desired level. They are then transduced with a viral vector carrying the desired gene cassette, and then the CAR T cells are expanded to the level of one billion cells. Lentiviral vectors have been shown to efficiently transduce T cells and are therefore preferred vectors for introducing CARs into target cells in patients. The expanded cells are then reintroduced into the patient.

In certain in vivo applications, a nucleic acid encoding a fusion protein described herein or a vector comprising a nucleic acid encoding a fusion protein described herein is administered to an individual in need thereof. For example, a recombinant expression vector comprising a nucleic acid encoding a fusion protein described herein may be provided as described in, e.g., Nawaz et al., Blood Cancer Journal Vol. 11, Article Number: 119 (June 23, 2021 ); Carbonaro-Sarracino et al., Molecular Therapy: Methods & Clinical Development, Volume 16, March 2020; Cantore and Naldini Haemophilia, Volume 27, Issue S3, Pages 122-125; Gouze-Decaris et al., Arthritis Res 2001; 3(Suppl 1): p. 34; Breuer et al., Scientific Reports Volume 10, Article No. 4544 (2020), Naldini et al., SCIENCE • April 12, 1996 • Volume 272, Page 5259 Issue • Pages 263-267. Viral vector particles can be used to deliver nucleic acids directly in vivo. Examples of such viral vector particles include lentivirus, retrovirus, AAV, HVS, vaccinia virus, and many other virus types. Viral vector particles can be modified to optimize delivery, for example, to specific cell types, including immune cells. See, e.g., Ang et al., PNAS, 2006 Aug 1 | 103 (31) 11479-11484; Schaffer et al., Annu Rev Biomed Eng. 2008; 10: 169–194; Lee et al., J. of Controlled Release No. Volume 334, June 10, 2021, pages 106-113; Jang et al., Molecular Therapy Volume 19, Issue 8, August 2011, pages 1407-1415.

Recombinant expression vectors can be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al. Human, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Constructs of circular or linear expression vectors can be prepared to contain replication systems that function in prokaryotic or eukaryotic host cells. Replication systems may be derived from, for example, ColEl, 2μ plasmid, lambda, SV40, bovine papillomavirus, etc.

Recombinant expression vectors may include one or more marker genes that allow selection of transformed or transfected hosts. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc.; provide prototrophic complementation in auxotrophic hosts; and the like. Suitable marker genes for use in recombinant expression vectors include, for example, neomycin/G418 resistance gene, puromycin resistance gene, hygromycin resistance gene, histidinol resistance gene, tetracycline resistance gene, and ampicillin resistance gene. sex genes.

Vectors useful in the context of the present invention may be "naked" nucleic acid vectors (ie, vectors with little or no proteins, sugars and/or lipids surrounding them), or vectors complexed with other molecules. Other molecules that may be suitably combined with the carrier include, but are not limited to, viral capsids, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties such as ligands, receptors, or antibodies that target cellular molecules.

The vector DNA can be introduced into cells, such as immune cells (eg, T cells) via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" and "transduction" are intended to refer to a variety of techniques recognized in the art for introducing foreign nucleic acids (e.g., DNA) into cells, including calcium phosphate or chloride. Calcium co-precipitation, DEAE-polydextrose-mediated transfection, lipofection, gene gun or electroporation. The cell lines used to produce viral vector particles themselves can be modified to introduce useful characteristics, including, but not limited to, non-self-shielding peptides, cell-type trophic peptides, anti-immunosuppressive peptides, and half-life extending peptides. protein therapeutics

In some embodiments, a fusion protein described herein can be produced as a protein therapeutic and administered to an individual instead of or together with a cell therapeutic described herein. Such polypeptides can be included in compositions, such as pharmaceutical compositions, and used as protein therapeutics. For example, protein therapeutics including fusion proteins described herein can be administered in combination with CD19-targeting cell therapeutics, such as CAR-T cells or ADCs.

A variety of methods for preparing polypeptides are known in the art and can be used to prepare polypeptides for inclusion in protein therapeutics. For example, a polypeptide can be produced recombinantly by utilizing a host cell system engineered to express a nucleic acid encoding the polypeptide. Recombinant expression of genes may include construction of expression vectors containing polynucleotides encoding polypeptides. Once the polynucleotide is obtained, vectors for producing the polypeptide can be generated by recombinant DNA technology using techniques known in the art. Known methods can be used to construct expression vectors containing polypeptide coding sequences and appropriate transcription and translation control signals. Such methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination.

The expression vector can be transferred to the host cell by conventional techniques, and the transfected cells can then be cultured by conventional techniques to produce the polypeptide.

A variety of host expression vector systems are available (see, eg, U.S. Patent No. 5,807,715). Such host expression systems can be used to produce polypeptides and subsequently purify them if desired. Such host expression systems include microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant phage DNA, plastid DNA, or myxoplasmic DNA expression vectors containing polypeptide coding sequences; recombinant yeast containing polypeptide coding sequences Yeast transformed with expression vectors (e.g., Saccharomyces cerevisiae and Pichia pastoris); Insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing polypeptide coding sequences; Insect cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic) Viruses, CaMV; Tobacco mosaic virus, TMV) infected plant cell systems or transformed with recombinant plastid expression vectors (e.g., Ti plastids) containing polypeptide coding sequences; or mammalian cell systems with recombinant expression constructs ( For example, COS, CHO, BHK, 293, NSO and 3T3 cells), these constructs contain promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., metallothionein promoters) For example, late adenovirus promoter; poxvirus 7.5K promoter).

For bacterial systems, a variety of expression vectors can be used, including but not limited to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791); pIN vector (Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109 ; Van Heeke and Schuster, 1989, J. Biol. Chem. 24:5503-5509); and its analogs. The pGEX vector can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).

For expression in mammalian host cells, virus-based expression systems can be used (see, eg, Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Expression efficiency can be improved by including appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see, eg, Bittner et al., 1987, Methods in Enzymol. 153:516-544).

Additionally, a host cell strain can be selected that modulates the expression of the inserted sequence or modifies and processes the gene product in the specific manner desired. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the polypeptides expressed. Such cells include, for example, established mammalian and insect cell lines, animal cells, fungal cells and yeast cells. Mammalian host cells include, for example, BALB/c mouse myeloma cell line (NSO/1, ECACC number: 85110503); human retinoblastoma cells (PER.C6, CruCell, Leiden, The Netherlands); SV40-transformed monkey kidney CV1 cells strain (COS-7, ATCC CRL 1651); human embryonic kidney cell strain (293 or 293 cell subculture for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977); Human fibrosarcoma cell lines (such as HT1080); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 , 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1 587); human cervical cancer cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44- 68, 1982); MRC 5 cells; FS4 cells; and human liver cancer cell line (Hep G2).

For long-term, high-yield production of recombinant proteins, host cells are engineered to stably express the polypeptide. Host cells can be transformed with DNA controlled by appropriate expression control elements known in the art, including promoters, enhancers, sequences, transcription terminators, polyadenylation sites and selectable markers. Methods generally known in the field of recombinant DNA technology can be used to select the desired recombinant pure line.

Once a protein described herein has been produced by recombinant expression, it can be purified by any purification method known in the art, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, and calibrated columns). chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. For example, antibodies can be isolated and purified by appropriate selection and combination of affinity columns (such as protein A columns) and chromatography columns, filtration, ultrafiltration, salting out, and dialysis procedures (see Antibodies: A Laboratory Manual, Ed Harlow , David Lane, Cold Spring Harbor Laboratory, 1988). Additionally, as described herein, polypeptides can be fused to heterologous polypeptide sequences to facilitate purification. Alternatively or additionally, the polypeptide or fusion protein may be prepared in part or entirely by chemical synthesis. viral delivery

In some embodiments, nucleic acids encoding fusion proteins described herein can be introduced into cells and/or administered to individuals as viral vectors. In some embodiments, such viral vectors can be used to introduce fusion proteins into cancer cells (eg, tumor cells). Introduction of such fusion proteins may increase susceptibility to an individual's immune system and/or one or more additional therapeutic agents (see, eg, WO2017/075533). carrier design

Nucleic acid sequences encoding the fusion proteins described herein can be introduced into a variety of types of vectors. For example, nucleic acids can be cloned into plastids, phagemids, phage derivatives, animal viruses, and myxoplasts. Other vectors may include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and viral vectors. In other examples, the vector may be a foamy virus (FV) vector, a retroviral vector made from a foamy virus. Viral vector design and techniques are well known in the art, as described in Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2001) and other virology and molecular biology manuals. viral transduction

Viruses are highly efficient at delivering nucleic acids to specific cell types, while often avoiding detection by the infected host's immune system. These characteristics make certain viruses attractive candidate vehicles for introducing cell therapy targets into cancer cells, such as solid tumor cells. A number of virus-based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, lentiviruses, poxviruses, herpes simplex virus type 1, herpesviruses, oncogenic viruses (e.g., murine leukemia virus), and Similar. Typically, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites and one or more selectable markers (e.g., WO 01/96584; WO 01/ 29058; and U.S. Patent No. 6,326,193).

Lentiviral and retroviral transduction can be enhanced by adding LentiBOOST (Mayflower Bioscience SBPLV10112) or TransDux (System Biosciences LV850 A-1) or polybrene (SantaCruz sc-134220; Millipore TR-1003-G; Sigma 107689) , Polybrene is a cationic polymer (also known as hexadimethrine bromide) used to improve the transduction efficiency of lentivirus or retrovirus.

For example, retroviruses provide a platform for gene delivery systems. Retroviruses are enveloped viruses belonging to the family Retroviridae. Once inside the host cell, the virus replicates by using viral reverse transcriptase to transcribe its RNA into DNA. Retroviral DNA replicates as part of the host genome and is called a provirus. Selected genes can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of the individual in vivo. Many retroviral systems are known in the art (see, eg, U.S. Patent Nos. 5,994,136, 6,165,782, and 6,428,953).

Retroviruses include alpharetroviruses (such as avian leukemia virus), betaretroviruses (such as mouse mammary tumor virus), deltaretroviruses (such as bovine leukemia virus and human T-lymphotropic virus), epsilon Retroviruses (eg, leukoderma sarcoma virus) and lentiviruses. In some embodiments, the retrovirus is a lentivirus, a genus of virus in the family Retroviridae that is, for example, characterized by a long latency period. Lentiviruses are the only retroviruses that can infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells and therefore can be used as efficient gene delivery vectors. In some examples, the lentivirus can be, but is not limited to, human immunodeficiency virus (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIA) and visna virus. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

In some embodiments, the vector is an adenoviral vector. Adenoviruses are a large family of viruses containing double-stranded DNA. It copies the host cell's DNA and uses the host's cellular machinery to synthesize viral RNA DNA and proteins. Adenoviruses are known in the art to affect both replicating and non-replicating cells, to accommodate large transgenes, and to encode proteins without integrating into the host cell genome.

In some embodiments, AAVP vectors are used. AAVP vector system is a hybrid of prokaryotic-eukaryotic vectors. These vectors are chimeras of genetic cis-elements of recombinant adeno-associated virus (AAV) and bacteriophage. AAVP combines selected elements of the phage and AAV vector systems to provide a vector that is easily produced in bacteria and exhibits few or no packaging limitations, while allowing infection of mammalian cells and integration into the host chromosome. Vector systems containing a number of appropriate elements are commercially available and can be further modified by standard methods to include the necessary sequences. Furthermore, AAVP does not require helper viruses or heterologous agents. Additionally, the absence of AAV capsid formation eliminates AAV's natural tropism for mammalian cells. Additional methods and details are found in US Patent 8,470,528 and Hajitou A. et al., Cell, 125: 358-398.

In some embodiments, human papilloma (HPV) pseudovirus is used. DNA plasmids can be packaged into papillomavirus L1 and L2 capsid proteins to generate pseudovirions capable of efficient DNA delivery. Encapsulation protects DNA from nucleases and provides targeted delivery with a high level of stability. HPV pseudoviruses can alleviate many of the safety concerns associated with the use of viral vectors. Additional methods and examples are found in Hung, C. et al., Plos One, 7:7(e40983); 2012, U.S. Patent 8,394,411, and Kines, R. et al., Int J of Cancer, 2015.

In some embodiments, oncolytic viruses are used. Oncolytic virotherapy selectively replicates the virus in cancer cells and can subsequently spread within the tumor, for example, without affecting normal tissue. Alternatively, oncolytic viruses can preferentially infect and kill cells without causing damage to normal tissue. Oncolytic viruses are also effective in inducing immune responses against themselves as well as against infected tumor cells. Generally, oncolytic viruses are divided into two categories: (I) viruses that preferentially replicate naturally in cancer cells and are not pathogenic to humans. Exemplary Class (I) oncolytic viruses include autonomous parvovirus, myxoma virus (poxvirus), Newcastle disease virus (NDV; paramyxovirus), reovirus, and Seneca valley virus (picornavirus). Category II (II) includes viruses that have been genetically manipulated for use as vaccine vectors, including measles viruses (paramyxoviruses), polioviruses (picornaviruses) and poxviruses (poxviruses). In addition, oncolytic viruses can include viruses that have been genetically engineered to have mutations/deletions in genes required for replication in normal cells rather than cancer cells, including adenovirus, herpes simplex virus, and vesicular stomatitis virus. Oncolytic viruses are useful as viral transduction methods due to their low probability of genetic resistance, as they can target multiple pathways and replicate in a tumor-selective manner. Due to in situ viral amplification, the intratumoral viral dose increases over time (compared to a decrease over time with small molecule therapies) and can have built-in safety features (i.e., drug and immune sensitivities). Preparations and administration

Certain embodiments of the invention include, for example, administering to a subject a cell therapeutic agent (or a population thereof) described herein, a protein therapeutic agent described herein, a composition comprising a cell therapeutic agent, and/or Methods of compositions containing protein therapeutics. In some embodiments, the method is effective in treating cancer in an individual.

In some embodiments, the cell therapeutic agent includes autologous cells that are administered to the same individual from whom the immune cells were obtained. Alternatively, immune cells are obtained from an individual and transformed, eg, transduced, with an expression construct described herein to obtain a cell therapeutic agent that is allogeneically transferred into another individual.

In some embodiments, the cell therapeutic agent is autologous to an individual, and the individual may be naïve, immune, diseased, or in another state prior to isolation of immune cells from the individual.

In some embodiments, additional steps may be performed prior to administration to an individual. For example, a cell therapeutic can be expanded ex vivo after immune cells are contacted (eg, transduced or transfected) with an expression construct described herein but before administered to an individual. In vitro expansion can be performed for 1 day or more, such as 2 days or more, 3 days or more, 4 days or more, 6 days or more, or 8 days or more. Alternatively or additionally, ex vivo expansion can be performed for 21 days or less, such as 18 days or less, 16 days or less, 14 days or less, 10 days before administration to the subject or less, 7 days or less, or 5 days or less. For example, in vitro expansion can be performed for 1-7 days, 2-10 days, 3-5 days, or 8-14 days prior to administration to an individual.

In some embodiments, the cell therapeutic may be stimulated with an antigen (eg, a TCR antigen) during ex vivo expansion. Antigen-specific amplification may optionally be supplemented by amplification under conditions that non-specifically stimulate lymphocyte proliferation, such as anti-CD3 antibodies, anti-Tac antibodies, anti-CD28 antibodies or phytohemagglutinin (PHA). The expanded cell therapeutic can be administered directly to the subject or can be frozen for future use, ie, for subsequent administration to the subject.

In some embodiments, the cell therapeutic agent is treated ex vivo with interleukin-2 (IL-2) prior to infusion into the cancer patient, and the cancer patient is treated with IL-2 after infusion. Additionally, in some embodiments, cancer patients may undergo preparative lymphocyte depletion (temporary ablation of the immune system) prior to administration of the cell therapeutic agent. The combination of IL-2 therapy and preparative lymphocyte depletion may enhance the persistence of cell therapeutic agents.

In some embodiments, a cell therapeutic is transduced or transfected with a nucleic acid encoding an interleukin, which can be engineered to provide constitutive, regulatable, or temporally controlled expression of the interleukin. Suitable interleukins include, for example, interleukins that act to enhance the survival of T lymphocytes during systole, which may promote the formation and survival of memory T lymphocytes.

In certain embodiments, the cell therapeutic agent is administered before, substantially simultaneously with, or after another therapeutic agent, such as a cancer therapeutic agent. Cancer therapeutic agents may be, for example, chemotherapeutic agents, biological agents, or radiation therapy. In some embodiments, the subject receiving the cell therapeutic agent is not administered treatment sufficient to cause immune cell depletion, such as lymphodepleting chemotherapy or radiation therapy.

The cell therapy agents described herein can be formed into compositions, such as cell therapy agents and pharmaceutically acceptable carriers. In certain embodiments, the composition is a pharmaceutical composition comprising at least one cell therapy agent described herein and a pharmaceutically acceptable carrier, diluent and/or excipient. Pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients and diluents, are well known and readily available to those skilled in the art. Preferably, the pharmaceutically acceptable carrier is chemically inert toward the active agent, such as the cell therapy agent, and does not cause any deleterious side effects or toxicity under the conditions of use.

The compositions may be formulated for administration by any suitable route, such as intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, and/or subcutaneous administration. Preferably, the composition is formulated for parenteral administration.

Compositions suitable for parenteral administration may be aqueous or non-aqueous isotonic sterile injectable solutions, which may contain, for example, antioxidants, buffers, bacteriostatic agents and solutes that render the composition isotonic to the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions may contain one or more suspending agents, solubilizers, thickeners, stabilizers and preservatives.

The dosage administered to an individual, particularly a human, will vary depending on the particular embodiment, the compositions used, the method of administration, and the particular site and individual being treated. However, the dose should be sufficient to provide a therapeutic response. A clinician skilled in the art can determine a therapeutically effective amount of a composition to be administered to a human or other subject to treat or prevent a particular medical disorder. The precise amount of a composition required for therapeutic efficacy will depend on many factors, such as the specific activity of the cell therapeutic, and the route of administration, on the tumor cells (e.g., as a result of tumor volume or tumor burden), and/or on normal cells. the amount of one or more available antigens, as well as a number of individual-specific considerations well known to those skilled in the art. In some embodiments, appropriate doses of cell therapeutic agents for one or more specific cancer indications can be defined in dose escalation clinical trials.

Any suitable number of cell therapy cells can be administered to an individual. Although single cell therapy cells described herein are capable of expanding and providing therapeutic benefit, in some embodiments, ¹⁰ or more, e.g., ¹⁰ or more, ¹⁰ or more, ¹⁰ or more, or 10 ⁸ or more cell therapy cells. Alternatively or additionally, the individual is administered ¹⁰ or less, such as 10 ^or less, ¹⁰ or less, ¹⁰ or less, or ¹⁰ or less as described herein. Cell therapy cells. In some embodiments, 10 ² -10 ⁵ , 10 ⁴ -10 ⁷ , 10 ³ -10 ⁹ , or 10 ⁵ -10 ¹⁰ cell therapy cells described herein are administered.

Doses of cell therapy agents described herein may be administered to the mammal once or in a series of doses over a suitable period of time, such as daily, biweekly, weekly, biweekly, semimonthly, every two Monthly, semi-annually or annually, depending on need. Dosage units containing an effective amount of the cell therapy agent may be administered in a single daily dose, or the total daily dose may be administered in two, three, four, or more divided doses per day, as desired.

The polypeptides described herein may be incorporated into pharmaceutical compositions (eg, used as protein therapeutics). Pharmaceutical compositions containing polypeptides may be formulated by methods known to those skilled in the art (see, for example, Remington’s Pharmaceutical Sciences, pp. 1447-1676 (ed. Alfonso R. Gennaro, 19th ed. 1995)). Pharmaceutical compositions may be administered parenterally in the form of injectable formulations containing sterile solutions or suspensions in water or another pharmaceutically acceptable liquid. For example, pharmaceutical compositions can be formulated by combining the polypeptide with a pharmaceutically acceptable vehicle or medium, such as sterile water and physiological saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, etc. The appropriate combination of excipients, diluents, vehicles, preservatives, and binders is then mixed in the unit dosage form required by generally accepted pharmaceutical practice. The active ingredient is included in the pharmaceutical preparation in such an amount as to provide a suitable dosage within the specified range.

Sterile compositions for injection may be formulated in accordance with common pharmaceutical practice using distilled water for injection as the vehicle. For example, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol and sodium chloride can be used as an aqueous solution for injection, with appropriate solubilizers as appropriate. , for example alcohols such as ethanol and polyols such as propylene glycol or polyethylene glycol, and non-ionic surfactants such as Polysorbate 80™, HCO-50 and similar combinations.

Non-limiting examples of oily liquids include sesame oil and soybean oil, and this may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are buffers such as phosphate buffer or sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The prepared injection may be packaged in suitable ampoules.

Protein half-life can be affected by the degree of sialylation, the post-translational covalent addition of terminal sialic acid to glycosylated proteins (Hossler et al., Glycobiology. 2009; 19:936–49). Proteins that are more sialylated may have longer half-lives in vivo (Flintegaard et al., Endocrinology. 2010; 151:5326–36; Bork et al., J Pharm Sci. 2009; 98:3499–508). In some embodiments, the fusion proteins described herein are formulated as sialylated protein therapeutics.

The route of administration may be parenteral, such as by injection, nasal administration, pulmonary administration, or transdermal administration. It can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

The appropriate investment method can be selected based on the age and situation of the individual. The single dose of the pharmaceutical composition containing the polypeptide can be selected from the range of 0.001 to 1000 mg/kg body weight. On the other hand, the dosage may be selected from the range of 0.001 to 100000 mg/kg body weight, but the present invention is not limited to such range. The dosage and method of administration may vary depending on the individual's weight, age, condition, and the like, and may be appropriately selected according to need by those skilled in the art.

In some embodiments, a pharmaceutical composition containing a fusion protein is administered in combination with a cell therapeutic agent, such as a CAR-T cell, or in combination with an ADC targeting CD19, as described herein. In some embodiments, a pharmaceutical composition includes a fusion protein described herein and a cell therapeutic agent described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered simultaneously, concomitantly, or sequentially with a cell therapeutic agent described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days prior to administration of a cell therapeutic agent described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 week, 2 weeks, 3 weeks, or 4 weeks prior to administration of a cell therapeutic agent described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 month, 2 months, 3 months, 4 months, or 5 months prior to administration of a cell therapeutic agent described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of a cell therapeutic agent described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 week, 2 weeks, 3 weeks, or 4 weeks after administration of a cell therapeutic agent described herein. In some embodiments, a pharmaceutical composition containing a fusion protein is administered about 1 month, 2 months, 3 months, 4 months, or 5 months after administration of a cell therapeutic agent described herein.

All publications, patent applications, patents and other references mentioned in this article, including GenBank accession numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

The invention is further illustrated by the following examples. These examples are for illustrative purposes only and should not be construed as limiting the scope or content of the invention in any way. CAR-T therapy relapse

Typically, CAR-T therapy involves the administration of T cells expressing chimeric antigen receptors (CARs) that bind an antigen of interest. In some embodiments, the CAR-T target antigen is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA) as described herein.

In some embodiments, the present invention is based in part on the recognition that certain individuals whose cancer relapses (e.g., ceases to exhibit one or more beneficial responses to CAR-T therapy, as described herein) after being treated with CAR-T therapy Relapse can be "rescued" by administration of a fusion protein described herein. In some embodiments, the invention provides compositions and methods comprising fusion proteins for the treatment of individuals exhibiting cancer recurrence during or following CAR-T therapy.

In some embodiments, the present invention is based in part on the recognition that certain individuals whose cancer is being treated with CAR-T therapy will have a suboptimal response to the therapy, and therefore may relapse, and therefore receive treatment to prevent relapse. As a non-limiting example, suboptimal responses can be improved by increasing the density of target antigen on tumor cells. In some embodiments, increasing the target antigen density on tumor cells can be achieved by binding the fusion protein of the invention to tumor cells. In some embodiments, the invention provides compositions and methods comprising fusion proteins for the treatment of individuals expected to have or have a suboptimal response to CAR T cell therapy, e.g., having achieved stable disease, partial response, excellent partial response or patients who had a complete response but did not achieve minimal residual disease negative status. (See eg www.cibmtr.org/manuals/fim/1/en/topic/multiple-myeloma-response-criteria). Individual identification

In some embodiments, individuals are identified and/or selected for administration of a fusion protein as described herein. In some embodiments, individuals can be identified and/or selected for treatment based on diagnosis of refractory or drug-resistant cancer. In some embodiments, individuals may be identified and/or selected for treatment based on prescription to receive ACT therapy. In some embodiments, individuals can be identified and/or selected for treatment based on evidence of relapse on ACT therapy. In some embodiments, identification and/or selection may be based on one or more measured or observed signs of cancer recurrence (e.g., non-beneficial response, loss or downregulation of target antigen of cells used in ACT, or progressive disease) Individual treatment. In some embodiments, the fusion protein is administered to the individual. In some embodiments, following administration of fusion protein therapy, the subject exhibits a positive clinical response to ACT therapy, e.g., exhibits improvement based on one or more clinical and/or objective criteria (e.g., exhibits tumor burden, tumor size and/or tumor stage).

Methods described herein may include preparing and/or providing reports, such as in electronic, web-based, or paper-based forms. The report may include one or more outputs from the methods described herein, such as tumor burden, tumor size and/or tumor stage, disease stability, loss or downregulation of the target antigen. In some embodiments, a report is generated, such as in paper or electronic form, that identifies the presence or absence of one or more tumor antigens in a cancer patient and, optionally, a recommended course of cancer therapy. In some embodiments, the report includes an identifier of the cancer patient. In one embodiment, the reporting is in a web-based form.

In some embodiments, additionally or alternatively, the report includes information regarding prognosis, resistance, or potential or suggested treatment options. The report may include information regarding the likely effectiveness of the treatment option, the acceptability of the treatment option, or the desirability of applying the treatment option to cancer patients, such as identified in the report. For example, the report may include information or recommendations regarding the administration of cancer therapy, such as administering to the patient a preselected dose or in a preselected treatment regimen, such as in combination with one or more alternative cancer therapies. The report may be delivered within 7, 14, 21, 30 or 45 days after execution of the methods described herein, for example, to the entities described herein. In some embodiments, the report is a personalized cancer treatment report.

In some embodiments, a report is generated for recording each time an individual is tested for cancer using the methods described herein. Individuals with cancer may be re-evaluated at regular intervals, such as monthly, every two months, every six months, or annually, or more or less frequently, to monitor the individual's response to cancer therapy and/or one or more cancers Improvement of symptoms, such as those described in this article. In some embodiments, the report may document at least the treatment history of the cancer individual.

In one embodiment, the method also includes providing the report to the other party. The other party may be, for example, the individual with cancer, a caregiver, a physician, an oncologist, a hospital, a clinic, a third-party payer, an insurance company, or a government agency. Examples Example 1. Construction and performance of CTE1 and CTE2 fusion proteins

The genetic fusion protein construct including secretion signal peptide, anti-CD20 VHH, part of CD19 ECD and anti-albumin llama VHH was selected into a mammalian cell expression vector and transiently transfected into Expi293 cells or CHO cells. The cell culture was clarified by centrifugation, and the fusion protein was purified from the supernatant. Two fusion proteins are produced. CTE1 (having the amino acid sequence of SEQ ID NO:2) is expressed in Expi293 cells. CTE2 (having the amino acid sequence of SEQ ID NO: 2 and lacking the C-terminal hexahistidine tag (e.g., SEQ ID NO. 55)) is expressed in CHO cells.

CTE1 fusion protein was purified by His-NTA column chromatography and analyzed by reducing and non-reducing PAGE. Superdex 75 purification was used to isolate the main peak and remove any endotoxin, and SEC-HPLC was used to separate the monomeric protein from the small aggregates present. CTE2 fusion protein was purified by protein A affinity chromatography, cation exchange chromatography and SEC-HPLC purification. The CTE2 fusion protein was purified to homogeneity as determined by SEC-HPLC and PAGE. Example 2. Biophysical characterization of CTE1 and CTE2 fusion proteins

Capillary isoelectric focusing (cIEF) analysis was used to assess sialylation of CTE1 and CTE2 fusion proteins. In cIEF separation, a continuous pH gradient created by applying a voltage across a capillary filled with carrier ampholyte separates proteins by their isoelectric point (pi). The pI of a protein is the pH value at which the total charge of the protein is zero. Thus, cIEF separates protein isoforms relative to total charge, which in turn is affected by the presence or absence of sialic acid relative to fusion protein isoforms. Briefly, proteins can be evaluated as the percentage of basic and acidic peaks, with the acidic peak representing sialylated protein.

Three protein preparations were evaluated for their degree of sialylation: CTE1 and CTE2 as described in Example 1, and CTE3 (SEQ ID NO. 55), a 100% purified from the second batch of CTE2 found to have sialylated and unsialylated forms. sialylated form. CTE3 was prepared by separation using an anion exchange column (AEX) and collecting the eluate.

Sialylation was assessed using imaging capillary isoelectric focusing (icIEF), a high-resolution technique that separates species based on the inherent net charge of the pi of the molecule. icIEF takes into account surface exposure and internal amino acids, without loss of resolution due to hydrophobic interactions. icIEF separation is performed by focusing isoforms in an amphoteric pH gradient via application of an electric field. The focusing step is followed by the detection step, which requires chemical or pressure adjustment via a fixed UV detector in conventional capillary isoelectric focusing (cIEF). icIEF lacks an adjustment step, and detection is achieved by continuously scanning the entire capillary. Since the mobilization step is eliminated, charge variation patterns can be recorded with the highest possible resolution within an effective run time of 15–20 minutes. The protein preparations CTE1, CTE2 and CTE3 showed different icIEF profiles as shown in Figure 1. Specifically, CTE1 is a mixture of sialylated and non-sialylated proteins, CTE2 is almost entirely a non-sialylated protein, and CTE3 is almost entirely a sialylated protein.

Since the appearance of basic and acidic peaks may be affected by other characteristics besides sialylation, sialidase treatment was performed to demonstrate that the presence of acidic peaks is due to sialic acid modification within the protein preparation. Several CTE1 preparations in different formulation buffers (FB) were evaluated for +/- sialidase, these are coded as CTE-Test and CTE-Test 2 in Figure 2.

This experiment shows that sialidase removes all of the more acidic peaks (pH 5.1 – pH 7.9) and slightly reduces the size of one alkaline peak (pH 8.55). Example 3. Fusion protein binds CD20 and albumin

Binding of CTE1 and CTE2 fusion proteins (as described in Example 1) and CTE3 (AEX preparation) to CD19 positive and CD19 negative cells was assessed. Briefly, the JeKo-1 mantle cell lymphoma cell line was CRISPR/Cas9 edited to eliminate CD19 expression, thereby generating the JeKo-19KO cell line. JeKo-19KO cells express CD20 but not CD19. Cells were incubated with CTE proteins, washed to remove unbound protein, and then incubated with anti-CD19 antibody FMC63 labeled with phycoerythrin (FMC63-PE). The cells were then washed again and fixed with PBS containing 2% formalin for analysis by flow cytometry.

As shown in Figure 3, CTE1 (red circles), CTE2 (open green squares) and CTE3 (open blue circles) proteins bind equivalently to CD19-negative/CD20-positive JeKo-19KO cells, as shown by anti-CD19-PE antibody ( FMC63), that is, the degree of sialylation is not considered. The calculated half-maximum effective concentrations based on these combined data are reported in Table 1. Table 1: test molecule EC ₅₀ (ng/ml) EC ₅₀ (nM) CTE1 7.5 0.13 CTE2 3.2 0.06 CTE3 11.5 0.2

The fusion protein was evaluated for binding to CD20 and albumin. Briefly, two ELISA formats were performed using the anti-CD19 antibody FMC63 to capture the fusion protein. ELISA plates (Thermo Fisher) were coated with 1 µg/ml FMC63 overnight at 4°C and blocked with 200 µl/well of Tris-buffered saline (TBS) containing 0.3% skim milk for 1 hour at room temperature. The fusion protein was added to TBS containing 1% BSA at gradually decreasing concentrations and incubated for 1 hour. The plate was washed 3 times with TBS. Then, 0.5 µg/ml biotinylated human albumin (Novus Biologics) or 0.5 µg/ml biotinylated human CD20-"Nanodisc" (Acro Biosystems) was added to TBS containing 1% BSA and maintained for 1 hour. The plates were washed again and then incubated with streptavidin-HRP and TMB peroxidase substrate (Thermo Fisher) to detect bound proteins.

As shown in Figure 4, CTE1 (green triangle), CTE2 (red circle), and CTE3 (blue square) proteins bind equivalently to anti-CD19-coated disks, such as with biotinylated albumin (left) or biotin CD20 membrane preparation (right), that is, regardless of the degree of sialylation. The calculated half-maximum effective concentrations based on these combined data are reported in Table 2. Table 2: test molecule EC ₅₀ ng/ml ( biotinylated albumin ) EC ₅₀ ng/ml ( biotinylated CD20) CTE2 13.5 22.3 CTE1 24.6 22.0 CTE3 15.1 18.6 Example 4. Fusion protein mediates CAR-19 T cell targeting and cytotoxicity

CTE1 and CTE2 fusion proteins (as described in Example 1) were evaluated for CAR-19 T cell targeting and cytotoxicity. Briefly, 50 μL of JeKo-carrying luciferase gene was seeded in a 96-well circular bottom plate at a density of 1 × ¹⁰ cells/well in RPMI 1640 medium containing 10% FBS and without antibiotics (RPMI/FBS). 1 CD19KO target cells. Test proteins were diluted in 50 μL RPMI/FBS and added to cells. CAR-19 T cells were thawed and washed once with RPMI/FBS, collected via centrifugation at 550 RCF for 10 min and added to the wells in a volume of 50 μL to give defined CAR-19 T:target (E:T) cells Compare. The plates were incubated at 37°C for 48 hours. The plate was centrifuged at 550 RCF for 5 minutes, the pellet was rinsed with PBS, and centrifuged again. Next, 20 μL of 1× lysis buffer (Promega) was added to the pellet, and the lysate was transferred to a 96-well opaque tissue culture plate (Fisher Scientific). The plate was read in a photometer with a syringe to dispense substrate (Promega). Percent killing is calculated based on the average luminescence loss of experimental conditions relative to control conditions of target cells only plus CAR-19 T cells.

As shown in Figure 5, CAR-19 T cells have equivalent cytotoxic activity in the presence of target JeKo-19KO cells and CTE1 (red circle), CTE2 (blue square) and CTE3 (open triangle) proteins, and also That is, the degree of sialylation is not considered. The calculated half-maximal inhibitory concentrations based on these cytotoxicity data are reported in Table 3. table 3: CTE1 CTE2 CTE3 Cytotoxicity IC50 (ng/ml) 0.012 0.018 0.020

These results indicate that the CTE fusion protein can redirect CAR-19 T cells to CD19 that binds to and is displayed on the CD20 cell surface protein. Fusion proteins that bind to CD20 and display the extracellular domain of CD19 increase the apparent density of CD19 on wild-type lymphoma cells that naturally express both antigens. Using the “Bang Beads” method (Bangs Laboratories, Fishers, IN USA), when CTE3 was added to wild-type JeKo-1 mantle cell lymphoma cells or wild-type Ramos-Hodgkin lymphoma cells, lymphoma cell surface The number of apparent CD19 molecules above was shown to increase in a dose-dependent manner. The total number of measurements was found to be approximately equal to the number of CD19 receptors plus the number of CD20 receptors measured by flow cytometry staining. Experiments were performed using the manufacturer's instructions. method

Bead staining (Bangs Lab QSC beads catalog number: 815B5ML; anti-mouse IgG, lot number 15515, access code: 22020217-1) : Manually shake the bottle to ensure uniform suspension. Do not vortex. Blank population: no staining. Each marker population (1-4) was stained separately. Add one drop of QSC beads to 50 μL of FACS buffer and tap gently to mix. Add 30 μL FMC63-PE (for a total of 3 × ¹⁰ cells) for CD19 standard curve beads, add 60 μL a-CD20-PE (for a total of 3 × ¹⁰ cells), and tap gently to allow immediate mix. Incubate in the dark at 4°C for 30 minutes. Add 1 ml FACS buffer and centrifuge at 2500g for 5 minutes. Wash twice with 1 ml FACS buffer and centrifuge at 2500g for 5 minutes; resuspend in 500 μL PBS.

Cell staining: In a 96-well FACS plate, 50 μL of Fc-blocked cells (approximately 5×10 ^{5 cells} ) were mixed with 50 μL of 3× serially diluted 606 protein (CTE3, SEQ ID NO. 55, sialylated) at 4°C. Incubate for 30 minutes. Wash cells 2 times with 200 μL FACS buffer. Resuspend cells in 50 μL FACS buffer, add 30 μL FMC63-PE, and incubate at 4°C for 30 minutes. For controls: a. Unstained cells , b. FMC63-PE stained cells (30 μL/test, for 3×10 ⁶ cells), c. Anti-CD20-PE stained cells (60 μL anti-CD20-PE, for 3×10 ⁶ cells), Tap gently to mix immediately. Incubate cells in the dark for 30 minutes at 4°C. Wash cells 3 times with 200 μL FACS buffer and centrifuge at 500g for 2 minutes. Resuspend in 200 μL PBS containing 1% paraformaldehyde Resuspend cells to fix cells for 30 minutes. FACS analysis: Run beads and cells on the same day with the same settings.

Analyze beads on Accuri C6 plus machine: set flow rate to 100-200 events/second (media flow control) and collect 1000 events per bead population. Stained beads can be combined and run in a single tube (5×1000 events). Using the FSC/SSC dot plot, create FSC-H/FSC-A to obtain single peaks and SSC-A/FL2 (PE channel). Create a histogram/FL2 (PE channel) and apply half-height/full-width gates to each bead population (M1, M2, M3, M4, M5). Record the geometric mean or median FL2 (PE channel) for each bead population for entry into the QuickCal spreadsheet (use access code to download QuickCal v 3.0). Enter the median value of FL2 (PE channel) to obtain the standard curve.

Cells were run on Accuri C6 plus with the same settings as the bead analysis, collecting 5000 events per test. Using the FSC/SSC dot plot, create FSC-H/FSC-A to obtain single peaks and SSC-A/FL2 (PE channel). Create a histogram/FL2 (PE channel) and apply half-height/full-width gates to the cell population. Record the geometric mean or median FL2 (PE channel) of the cell population for entry into the QuickCal spreadsheet. Enter the FL2 (PE channel) median and obtain the ABC (antibody binding capacity) value. Subtract the ABC value of the unstained cell population or isotype control from the ABC value of the cells. If it is assumed that a monovalent antibody binds to a surface receptor, then the ABC value = # surface receptor. The results showed that the apparent number of CD19 molecules on the surface of JeKo-1 cells increased significantly in a dose-dependent manner in the range between 10 ng/ml and 10 µg/ml CTE3. The results showed that the apparent number of CD19 molecules on the surface of Ramos cells increased significantly in a dose-dependent manner in the range between 10 ng/ml and 10 µg/ml CTE3. The results showed that the effect of increasing the number of CD19 molecules on target lymphoma cells enhanced CAR-19-mediated toxicity for up to 24 hours at different E:T ratios. Results showed enhanced cytotoxicity over a range of E:T ratios including 0.1:1, 0.3:1, 1:1, 3:1 and 5:1.

It is concluded that CTE3 increases apparent CD19 density on lymphoma cells to a level sufficient to increase CAR-19-mediated cytotoxicity. Example 5. Using fusion proteins to treat CD19 antigen escape

CTE1 fusion proteins (as described in Example 1) were evaluated for their ability to modulate CD19 antigen escape from target cells. To generate the CD19 antigen escape model, wild-type CD19-positive/CD20-positive JeKo-1 cells carrying the luciferase gene (JeKo-luc) were seeded at 1 × 10 ⁴ cells in 100 μl per well of a 96-well circular bottom plate. in RPMI/FBS. 100 μl of RPMI/FBS containing CAR-19 T cells was added to give a JeKo-luc:CAR-19 T cell ratio of 1:1, 0.3:1, or 0.1:1, and the co-cultures were grown for up to 13 days. Samples were assessed for accumulation of CD19 ^- CD20 ⁺ populations (FMC63-PE, Millipore; anti-CD20-APC, BD Pharmingen) by flow cytometric analysis on days 1, 6, 11 and 13.

Once a CD19-negative population emerged, JeKo-luc cells were reseeded at 1 × 10 ⁴ cells after enumeration by flow cytometry using bead counting following the manufacturer's protocol (Bang Laboratories, Inc). in 50 μl RPMI/FBS. Next, 50 μl RPMI/FBS containing 1 × 10 CAR-19 T cells was added to give a JeKo-luc:CAR-19 T ratio of 10:1 ^, with or without fusion protein in 50 μl RPMI/FBS . In one set of control wells, the fusion protein alone was added to JeKo-luc cells without the addition of CAR-19 T cells. In each case, 150 μl of the co-culture was incubated for 48 hours. Next, cells were stained with HIB-CD19-PE (BioLegend) and anti-CD20-APC (BD Pharmingen) and samples incubated without protein or treated with protein were analyzed by flow cytometry for anti-CD20-APC. JeKo-1 cells cultured with anti-ROR1-PE (BioLegend) antibody. 7AAD is used to exclude dead cells.

Wild-type JeKo-1 cells have a very high proliferation index and are used as a model for antigen escape from CAR-T cell therapy. The phenotype of these cells under CAR-T stress was first characterized. JeKo-1 cells were CD19 positive and CD20 positive, and most of them were double positive, as shown in Figure 6 .

Figure 6 shows that wild-type JeKo-1 cells express CD19 (X-axis) and CD20 (Y-axis), so almost all cells (90%) are located in the upper right quadrant, indicating double positivity.

Use titration of the effector:target cell ratio (E:T, here CAR-T:JeKo-1), in which the number of CAR-T cells changes relative to a fixed number of JeKo-1 cells and is further varied by The duration of culture creates a clear model of antigen escape.

Figure 7 shows the development of the JeKo-1 antigen escape model after treatment with CAR-19 T cells in vitro. The locations of CAR-19 cells and target JeKo-1 cells are indicated in the flow cytometry map, shown on the left side of the figure. The names of the CD19-positive and CD20-positive cell populations are shown on the right side of the figure. A) From day 1 to day 13, the E:T ratio is 1:1. B) From day 1 to day 13, the E:T ratio is 0.3:1. C) From day 1 to day 13, the E:T ratio is 0.1:1.

As shown in Figure 7, JeKo-1 cells can evade CAR-19 cytotoxicity through loss of CD19 expression at the cell population level. As shown in Figure 7A, days 6 and 13, this occurs even if the E:T ratio is 1:1. As shown in Figure 7B (days 6 and 13) and Figure 7C, the effect was more obvious at 0.3:1 and 0.1:1 E:T ratios, with the effect being particularly obvious on day 13. Notably, the 0.1:1 E:T ratio was ineffective at the level of cytotoxicity readout in the luciferase assay, but the selective pressure exerted by the CAR induced a predominantly double-positive (CD19 and CD20-positive) population (Fig. 7C, pp. 1 day) to a mixed population (Figure 7C, day 6) to a population dominated by CD19-negative and CD20-positive cells (Figure 7C, day 13).

Because escape occurred within this range of E:T ratios and days of incubation, a second study was performed to evaluate the effect of adding a CD19-anti-CD20 fusion protein.

Figure 8A shows CD19 (x-axis) and CD20 (y-axis) expression levels after 13 days of incubation of CAR-19 cells with JeKo-1 cells (E:T 1:1), as measured by flow cytometric analysis. Test. The performance of CD19 and CD20 was monitored by FACS. Figure 8B shows the results when only CTE1 protein, only CAR-19 T cells, or both CTE1 and CAR-19 T cells were added to the culture.

In this experiment, the addition of CAR-19 T cells and CTE1, but not CAR-19 T cells or CTE1 protein alone, was sufficient to kill all target JeKo-1 cells after escape.

These results demonstrate that antigen escape can be induced in vitro, particularly from CD19 expression, and can be reversed using CTE1 targeting CD20. Example 6. Treatment of cancer in mice using fusion proteins

The tumor therapeutic efficacy of CTE1 and CTE2 fusion proteins (as described in Example 1) was evaluated in mouse models. Briefly, 6-10 week old female NOD- scid IL2Rγ ^null (NSG) mice (Jackson Labs) were obtained and acclimated in a vivarium for at least 3 days before the start of the study. All animals were socially housed in static, sterile biocontaining disposable cages prefilled with corncob bedding. Food (LabDiet) and acidified water were provided ad libitum. On study day 1, mice were intravenously injected with 2.5 × 10 ⁶ JeKo-19KO cells at 0.1 mL/animal. On study day 3, all animals were imaged and then randomized to give equivalent mean tumor burden in each of 6 different cohorts of n = 8 mice/cohort. The next day, study day 4, different groups of mice were given fusion protein at doses ranging from 0 to 5 mg/kg, depending on the study, followed 3-4 hours later by 1 × ¹⁰ CAR-19 T cells. The fusion protein was then administered three times a week for a total of 14 injections. Control groups were mice treated with CAR-19 T cells alone without protein injection, and untreated mice that received no treatment.

Animals were subjected to cageside health examinations and clinical observations at least once daily. If animals in the study exhibit abnormal clinical signs, clinical observations will be conducted more frequently. Body weights were recorded before tumor induction, CAR-19 T cell administration, and three times weekly thereafter, unless the animal's health indicated otherwise. The most recently collected body weight is used to calculate protein and D-luciferin dosage. Imaging was performed on day 3 to randomize animals into groups with similar mean baseline luminescence. Following infusion of CAR-19 cells and CTE protein, all animals underwent whole-body imaging twice weekly, 10-15 minutes after subcutaneous administration of 15 mg/mL luciferin in 0.2 mL PBS. Isoflurane is administered during this procedure. Values above 1×10 ¹⁰ total flux (measured in lumens) are at the maximum and are considered a reason for humane euthanasia.

Several studies were conducted using JeKo-19KO cell line, CTE protein and CAR-19 T cells. The animals were injected with JeKo-19KO lymphoma cells and allowed to engraft for four days before starting treatment with CAR-19 T cells and CTE1 protein. CAR-19 T cells were injected once, and CTE1 protein was given three times a week. Tumor burden was quantified using luciferase-mediated luminescence.

In the first study, the in vivo efficacy of the CTE1 protein was tested. A dose of 2.5 × 10 ⁶ JeKo-CD19-KO cell tumor cells was injected intravenously into NSG mice. On day 3, CAR-19 T cells (1×10 ⁷ ) and CTE1 were added. CTE1 was then administered three times weekly, but was stopped on day 32 to monitor for lymphoma recurrence. As shown in Figure 9, mice treated with anti-CD19 CAR T cells and CTE1 were cured, and no recurrence was found as of day 45. Included CAR-19 and CAR-CD20 cohorts without CTE1; as expected, CAR-19 treated animals developed lymphoma and 4 of 5 animals died by day 45, untransduced T cell controls The same is true for group (UTD). The CD20 CAR worked well, except for one animal that developed fatal systemic lymphoma.

These results indicate that mice treated with CAR-19 T cells plus CTE1 protein were protected from lethal lymphoma. In addition, even at the lowest dose used, 0.56 mg/kg, durable responses were achieved after discontinuation of the drug.

In the second study, CTE1 and CTE2 protein formulations were screened for activity at a 2 mg/kg protein dose compared to the CAR-19 T cell only group and the untreated group. As shown in Figure 10, three times weekly administration of CTE1 and CTE2 proteins inhibited lymphoma development until day 31, when untreated (NA) mice and CAR-19 treated mice began to succumb to the disease.

These results indicate that at a dose of 2 mg/kg, CTE1 and CTE2 proteins have similar effects on lymphoma growth in vivo.

In the third study, two parameters were further investigated. First, dose titration of CTE2 was performed in 4 cohorts: 2 mg/kg, 0.4 mg/kg, 0.08 mg/kg, and 0.016 mg/kg. Next, the animals were administered protein until day 31, at which time administration was discontinued to assess the animals for signs of lymphoma recurrence. Nine days later, on day 42, animals treated with CAR-19 T cells plus 2 mg/kg CTE2 had no signs of luminescence, and 2 of the 8 animals treated with CAR-19 T cells plus 0.4 mg/kg CTE2 were showing signs of luminescence. Only minimal signal is observed, as shown in Figure 11. Lower doses of CTE2 were less effective in preventing relapse after drug withdrawal.

These results indicate that administration of CAR-19 T cells and CTE2 prevents Lymphoma relapses.

Body weight is a quantitative means of tracking animal health and is often used in conjunction with clinical examination to ensure humane euthanasia in cancer models. Mice treated with CAR-19 plus CTE2 protein continued to gain weight throughout the protein dosing period (until day 31), whereas control animals began to lose weight at approximately day 25, as shown in Figure 12. Even after CTE2 protein administration was stopped, animals in the treatment group maintained body weight by the end of the study (day 42), while animals in the highest dose treatment group received 2 mg/kg of CTE2 until day 31, and at day 39 Continue to gain weight from the last recorded weight on the day.

These results indicate that animals in the three highest dose cohorts were healthy enough to gain weight even after CTE2 administration was stopped on day 31, while the negative control group (CAR-19 only and ‘no treatment’) was healthy enough on day 31. After 21 days, the weight dropped steadily.

Figure 13 shows the lumen intensity of each group, showing a rapid increase in the untreated group and a slightly delayed but still rapid increase in the CAR-19 treatment group. Line termination for the untreated and CAR-19 treated groups corresponds to the loss of all animals in these groups. Because the signal intensity in the control group was very high, differences between the CTE2-administered groups could not be discerned unless those groups were removed. In the absence of these groups, a clear dose response was evident due to the complete absence of luminescence signal in the 2 mg/kg CTE2 cohort as of day 42. Since the last protein dose was given on day 31, this indicates that these animals have eliminated CD19 negative lymphoma.

These results showed complete protection against lymphoma, as by all animals treated with 2 mg/kg CTE2 (8/8 animals showed no signal), most animals treated with 0.4 mg/kg CTE2 (6/8 Luminescence measured from 1 animal showing no signal) and half of the animals treated with 0.08 mg/kg CTE2 (4/8 animals showing no signal). The lack of luminescent signal and continued weight gain indicate that some mice are disease-free, an indication supported by Kaplan-Meier survival curves for different cohorts, as shown in Figure 14.

Survival data indicated that only 1 of 32 treated animals (3%) died of lymphoma during the study; this animal was in the lowest dose group of 0.016 mg./kg. In comparison, 14/16 (87.5%) control animals died of lymphoma during the study.

In vivo protein efficacy varies with the pharmacokinetic (PK) properties of the injected fusion protein. The PK properties of CTE1, CTE2 and CTE3 were evaluated in mouse serum after a single injection, as shown in Figure 15. The half-life of the fusion protein in mice depends on albumin-mediated recycling via FcRN binding because there is no antigenic target—the anti-CD20 domain does not bind mouse CD20 and the human CD19 ECD is not available in solution. Binding properties are measured, but the anti-albumin domain binds to mouse albumin. CTE1 protein was evaluated in the serum of Balb/c and NSG mice after a single IV injection and showed similar half-lives of 21 hours and 28 hours, respectively. The half-life of CTE2 (hyposialylated) in Balb/c mice was shorter than expected at 7.6 hours, and the half-life of CTE3 (hypersialylated) was the longest at 36 hours.

Protein half-life is also affected by the degree of sialylation, which is the post-translational covalent addition of terminal sialic acid to glycosylated proteins. Proteins that are more sialylated have longer half-lives in vivo . Table 4 summarizes the half-life of fusion proteins and icIEF profiles of fusion proteins in Balb/c mice and NSG mice. icIEF data show that the CTE1 protein contains a mixture of acidic and basic peaks, the CTE2 protein is almost completely alkaline, indicating the lowest degree of sialylation, and the CTE3 protein is almost completely acidic, indicating a high degree of sialylation. These results explain the different PK properties of the three protein formulations. Table 4 protein T _1/2 , Balb/c T _1/2 , NSG icIEF , alkaline pH peak % icIEF , acidic pH peak % CTE1 20.63 27.7 26.2 73.8 CTE2 7.6 nd 100 0 CTE3 36 nd 0 100 Example 7. Generation and testing of additional fusion proteins

Other fusion proteins were evaluated for their ability to bind and kill CD19-CD20+ Jeko-1 cells. First, anti-CD20 VHHs (SEQ ID NO:37-42) differing in CDR2 sequence within SEQ ID 6 were evaluated for their ability to bind CD20 on CD19- CD20+ Jeko-1 cells using FACS. The following table describes changes made to the CDR2 sequence of SEQ ID NO:37. As shown in Table 5, VHHs including the indicated CDR2 sequences showed varying levels of binding, from complete loss of activity to almost no loss of activity, as well as changes in mean fluorescence intensity (MFI). table 5: CDR2 sequence Effect on CD20 binding ITYSGGS 100% combined I SW SGG WI No binding I SW SGGSP Combined, lower MFI ITYSGG WI 100% combined I SW SGGS T greatly reduced combination ITYSGGS T Combined, lower MFI

Substituting Ser-Trp (SW) for Thr-Tyr (TY) at the N terminus of CDR2 of SEQ ID NO:37 significantly affects binding. Surprisingly, the highly non-conservative substitution of the Ser-Pro (SP) sequence at the C-terminus of CDR2 of SEQ ID NO:37 with Trp-Ile (WI) did not affect binding. Additionally, mutations within the CDR3 of the anti-CD20 VHH of SEQ ID NO: 37 (AA N PTYGSDWNAEN to AA D PTYGSDWNAEN) have only a modest effect on binding to CD20.

A series of fusion protein constructs were generated, including secretion signal peptide, anti-CD20 VHH, GGGGSGGGGSGGGGSGGGGS linker, part of the CD19 ECD, SRGGGGSGGGGSGGGGS linker, anti-albumin VHH, and a hexahistidine tag. The amino acid sequences of the constructs are provided in Table 6: Table 6: Structure number SEQ ID NO: 526 (CTE1) 6 631 10 632 14 633 18 634 twenty two 635 26 636 30 637 34

The fusion protein was purified and then analyzed for binding and killing of CD20-positive Jeko-1 CD19KO cells. The combination is summarized in Table 7 below: Table 7. 526 (CTE1) 631 632 633 634 635 636 637 EC ₅₀ (ng/ml) 23.13 2263 181 40.72 4401 115.9 13.94 45.05 EC ₅₀ (nM) 0.41 - 3.21 0.72 - 2.06 0.25 0.8 MW (kDa) 56.4 56.4 56.4 56.4 56.4 56.4 56.4 56.4

The results of direct cell binding of the construct to CD20 on JeKo-19KO cells, measured by binding of the anti-CD19 mAb FMC63, fully confirmed the preliminary FACS results. Constructs #631 and #634, with four and three AA changes (relative to SEQ ID NO:37) respectively, did not bind cells at all. Construct #633, with two AA changes relative to SEQ ID NO:37, is nearly equivalent to construct #526 (CTE1). Construct #635, which has a conservative change in the C terminus (Pro to Thr) (relative to SEQ ID NO:37), significantly loses five-fold binding; and construct #636 (relative to SEQ ID NO:37, CDR3 Asn mutated to Asp ) retains full activity. Importantly, combining the mutations in CDR3 with the two AA substitutions in construct #633 resulted in a construct (#637) that is nearly equivalent to construct #526. The binding curve is shown in Figure 16.

The killing effect of the fusion protein on JeKo-19KO cells in the presence of CAR-19 was evaluated. The information is summarized in Figure 17 and Table 8 below. Table 8: EC50 ng/ml EC50 pM MW kDa #526 (CTE1) 0.063 1.13 56 #631 6.59 117.68 56 #632 0.51 9.11 56 #633 0.14 2.5 56 #634 1.42 25.36 56 #635 0.32 5.71 56 #636 0.063 1.13 56 #637 0.24 4.29 56

Figure 17 and Table 8 show that the cytotoxicity data are consistent with the binding data. Example 8. Evaluating additional fusion proteins

An additional fusion protein construct #607 was generated containing a different anti-CD20 VHH. The amino acid sequences of the constructs are provided in Table 9: Table 9: Structure number SEQ ID NO: 526 (CTE1) 6 606 (CTE2) 2, but lacks the C-terminal hexahistidine tag (e.g., SEQ ID NO. 55) 607 50

Constructs #526 (CTE1) and #607 bound to cell surface CD20 almost equally, approximately 0.4 nM for construct #526 (CTE1) and approximately 0.8 nM for construct #607 (Figure 18, left panel). However, construct #607 was approximately ten times less effective at killing JeKo-19KO cells (Figure 18, right panel). Table 10: Binding (EC ₅₀ ) Cytotoxicity (IC ₅₀ ) construct MW ng/ml nM ng/ml pM #526 (CTE1) 56.3 25 0.44 0.078 1.39 #606 (CTE2) 56.3 19.3 0.34 0.053 0.95 #607 56.9 44.8 0.79 0.66 11.6 Example 9 : Biophysical characterization and optimization of biologics

Analyze the biophysical properties of the biologics described herein. Fusion proteins described herein are evaluated using standard techniques known in the art to evaluate the characteristics of biologics for use in therapy. The fusion protein (eg, as described in this example) is assessed for stability (eg, stability in composition buffers and/or biological fluids (eg, blood or saliva)).

The fusion proteins described in this example are formulated to exhibit optimal melting temperature (Tm), enhanced stability, reduced levels of aggregation and/or degradation, increased levels of protease resistance, and increased antioxidant levels in vivo. , formulations with enhanced biodistribution and/or improved PK/PD properties. Such characteristics are evaluated using standard methods and assays known in the art, such as Differential Scanning Calorimetry (DSC), Differential Scanning Fluorescence (DSF), Circular Dichroism (CD), Temperature Scanning Viscometer , analytical ultracentrifugation (AUC), size exclusion chromatography (SEC, SEC-MALS), dynamic light scattering (DLS), photoresist, zeta potential, capillary electrophoresis CE (e.g., CZE, MECC, gel CE, cIEF /iCIEF), gel electrophoresis (eg, native, SDS-PAGE, IEF), electron microscopy (eg, TEM, SEM). Formulation parameters that confer these properties include, for example, pH, osmolality, buffers (e.g., phosphates, acetates, and histidine), tonicity agents/stabilizers (e.g., sugars such as sucrose, trehalose, or mannose). Alcohols; polyols, such as sorbitol), bulking agents (e.g., lyoprotectants, such as mannitol), surfactants (e.g., polysorbates), antioxidants (e.g., methionine), metal ions /Chelating agents (such as ethylenediaminetetraacetic acid, EDTA) and/or preservatives (such as benzyl alcohol). Example 10. Fusion protein-mediated CAR-19 T cell targeting and cytotoxicity.

This example further demonstrates that fusion proteins as described herein mediate CAR-19 T cell targeting in a concentration-dependent manner. Additionally, this example further demonstrates that fusion proteins as described herein mediate CAR-19 T cell cytotoxicity. These properties are inter alia described in Su et al. Oncoimmunology 2022, Volume 11, Issue 1, e2111904 (page 13), the entire contents of which are incorporated by reference.

CTE3 fusion proteins (as described in Example 1) were evaluated for CAR-19 T cell targeting and cytotoxicity. Briefly, 50 μL of JeKo-carrying luciferase gene was seeded in a 96-well circular bottom plate at a density of 1 × ¹⁰ cells/well in RPMI 1640 medium containing 10% FBS and without antibiotics (RPMI/FBS). 1 CD19KO target cells. Test proteins were diluted in 50 μL RPMI/FBS and added to cells. CAR-19 T cells were thawed and washed once with RPMI/FBS, collected via centrifugation at 550 RCF for 10 min and added to the wells in a volume of 50 μL to give defined CAR-19 T:target (E:T) cells Compare. The plates were incubated at 37°C for 48 hours. The plate was centrifuged at 550 RCF for 5 minutes, the pellet was rinsed with PBS, and centrifuged again. Next, 20 μL of 1× lysis buffer (Promega) was added to the pellet, and the lysate was transferred to a 96-well opaque tissue culture plate (Fisher Scientific). The plate was read in a photometer with a syringe to dispense substrate (Promega). Percent killing is calculated based on the average luminescence loss of experimental conditions relative to control conditions of target cells only plus CAR-19 T cells. In addition, the "Bang Beads" method (Bangs Laboratories, Fishers, IN USA) was used according to the manufacturer's instructions to identify JeKo-1 CD19KO target cells, wild-type JeKo-1 mantle cell lymphoma cells, or wild-type Ramos Hodge. The number of apparent CD19 molecules on the surface of King's lymphoma cells.

CTE fusion proteins redirect CAR-19 T cells to CD19 that binds to and is displayed on the CD20 cell surface protein. Fusion proteins that bind to CD20 and display the extracellular domain of CD19 increase the apparent density of CD19 on wild-type lymphoma cells that naturally express both antigens. As shown in Figures 19A-19C, when CTE3 fusion protein was added to JeKo-1 CD19KO target cells, wild-type JeKo-1 mantle cell lymphoma cells, or wild-type Ramos-Hodgkin's lymphoma cells, lymphoma The number of apparent CD19 molecules on the cell surface increased in a concentration-dependent manner. The total number of CD19 receptors or CD20 receptors measured on test cells after treatment with 10 µg/mL CTE3 was found to be approximately equal to the number of CD19 receptors plus the number of CD20 receptors.

CAR-19 T cells have cytotoxic activity in the presence of target JeKo-19KO cells and CTE protein. As shown in Figure 20 (left panel), after 18 hours, CAR-19 T cells were treated with JeKo-1 cells (E:T 3:1) treated with 0.01, 0.1, 1, or 10 µg/mL CTE protein. After incubation, it has an average cytotoxic activity of at least 60%. CAR-19 T cells had an average cytotoxic activity of at least 50% when incubated with JeKo-1 cells (E:T 3:1) treated with 0.001 µg/mL CTE protein. After 18 hours, CAR-19 T cells had an average of at least 40% cytotoxic activity after incubation with JeKo-1 cells (E:T 1:1) treated with 0.01, 0.1, 1, or 10 µg/mL CTE protein. CAR-19 T cells had an average cytotoxic activity of at least 20% when incubated with JeKo-1 cells (E:T 1:1) treated with 0.001 µg/mL CTE protein. After 18 hours, CAR-19 T cells had an average of approximately 30% cytotoxic activity after incubation with JeKo-1 cells (E:T 0.3:1) treated with 0.01, 0.1, 1, or 10 µg/mL CTE protein. CAR-19 T cells had an average cytotoxic activity of at least 10% after incubation with JeKo-1 cells (E:T 0.3:1) treated with 0.001 µg/mL CTE protein. After 48 hours, CAR-19 T cells showed higher cytotoxic activity after incubation with JeKo-1 cells.

These results further demonstrate that the fusion protein increases apparent CD19 density on lymphoma cells to a degree sufficient to increase CAR-19-mediated cytotoxicity. Example 11 : CTE3 binds to human and cynomolgus monkey CD20 expressed on 293T cells

This example further demonstrates that fusion proteins as described herein bind to human and cynomolgus monkey CD20.

Human 293T cells were grown to approximately 80% cell density. Cells were transfected with cDNA expressing human CD20 (GenScript OHu00965D) or "cynomolgus" CD20 (A157V mutation of human CD20 cDNA) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). The only amino acid difference between the extracellular domain of the A157V mutant line and human CD20. Approximately 48 hours after transfection, cells were removed by Accutase and harvested by centrifugation at 500xg for 2 minutes at 4°C. The cell pellet was resuspended in FACS buffer (PBS containing 1% BSA and 0.1% sodium azide). Next, 5 μl of Fc blocker (BD) per 10 ⁶ cells was added to the cell suspension and incubated at room temperature for 10 minutes. Cells were then centrifuged as above and resuspended in FACS buffer using 50 µl (approximately 5×10 ⁵ cells) for each sample. Next, 50 µl of CTE3 fusion protein was added to the cells starting from 10 µg/ml (final concentration) and followed by 3-fold serial dilutions in FACS buffer. The plate was incubated at 4°C for 30 minutes and then centrifuged as described above. The cell pellet was washed twice with FACS buffer and then resuspended in 50 µl of diluted FMC63-PE (2.5 µl per 50 µl of FACS buffer) and incubated at 4°C for 30 minutes. Cells were washed twice as above and then fixed with 150 µl 2% PFA. Samples were analyzed on a BD Accuri C6 flow cytometer using FlowJo software.

As shown in Figures 21A and 21B, CTE3 binds human and non-human primate (cynomolgus macaque) CD20 with an EC50 of approximately 6 ng/ml. These results indicate that CTE3 is cross-species specific between humans and non-human primates (cynomolgus macaques). Example 12 : CTE3 is active as a CAR T cell engager in the presence of human serum or human serum albumin

This example further demonstrates that CTE3 remains highly cytotoxic against CD20-expressing cells at pM concentrations.

CTE3 contains an albumin-binding domain. The ability to kill cells expressing CD20 in the presence of human serum or human serum albumin (HSA) was assessed. Cytotoxicity assays used luciferase-expressing JeKo-1 CD19KO-luc cells to determine CTE3 activity in the presence of 50% human serum or 30 or 60 mg/ml HSA. JeKo-1 CD19KO-luc cells were seeded at 1 × ¹⁰ cells in (96 round chassis) RPMI medium containing 50% human serum or 10% FBS (control condition) or without human serum albumin, containing 30 or 60 mg/l human serum albumin in 10% FBS RPMI medium, 50 µl/well. CTE3 was titrated in 4-fold serial dilutions starting at 100 ng/ml in the same serum/HSA dilutions as above. Dispense CTE3 dilution (25 µl) into the appropriate wells. 2:1 or 5:1 in RPMI medium containing 50% human serum or 10% FBS or in 10% FBS RPMI medium without human serum albumin containing 30 or 60 mg/ml human serum albumin The ratio of CAR19 T cells (CAR T cells to target cells) was added at a volume of 25 µl/well. The plates were incubated at 37°C for 48 hours. After the cells were washed and pelleted, lysis buffer (Glomax Multi Detection System) was added, the lysate was transferred to a 96 white opaque plate, and measured using a GloMax plate reader (Promega) with an auto-injector. Luciferase levels. Use GraphPad Prism software to draw data graphs. Table 11: CTE3 IC 50 ng/ml IC50 pM In 50% HS 0.46 8.3 In 10% FBS 0.11 2

As shown in Figure 22A, human serum reduced _{the IC50} of CTE3 approximately 4-fold compared to the IC50 of samples containing 10% FBS. IC50 values for the cytotoxicity assay were determined and shown in Table 11. CTE3 was less potent in 50% human serum (IC ₅₀ of approximately 8.3 pM) than in 10% FBS (IC ₅₀ of approximately 2 pM). In control wells containing CAR19 cells but no CTE3, reduced cell growth was observed in the presence of 50% human serum compared to 10% FBS. Table 12: CTE3 IC ₅₀ (ng/ml) IC ₅₀ pM In 10% FBS 0.09 1.62 In 30 mg/ml HSA 0.35 6.3 in 60 mg/ml HSA 0.18 3.2

As shown in Figure 22B, inclusion of HSA at 30 mg/ml or 60 mg/ml reduced the ability of CTE3 to induce cytotoxicity in JeKo-1 CD19 KO-luc cells compared to control samples containing 10% FBS. _IC50 values for the cytotoxicity assay were determined and shown in Table 12. In the presence of HSA, a 2-4-fold decrease in _IC50 was observed, and the maximum cytotoxicity level was slightly reduced compared to samples analyzed in 10% FBS only.

These results indicate that, although activity is reduced in the presence of 50% human serum or physiological levels of HSA, CTE3 is still highly cytotoxic against CD20-expressing cells at pM concentrations. Example 13 : CD20 x CD79b Bispecific Anti -CD19 CAR T Adapter Protein Antigen Binding

The bispecific CD79b x CD20 CAR19 adapter protein is generated by assembling anti-CD79b scFv with anti-CD20 VHH and CD19 ECD in CTE3. The constructs placed the anti-CD79b scFv at the N-terminus (#650) or C-terminus (#651) of the anti-CD20 VHH-CD19 ECD core sequence. Plasmids were transfected into 293T cells using Lipofectamine 2000, and supernatants were collected and titrated. Binding of #650 and #651 proteins to biotinylated CD20 or CD79b was analyzed in an ELISA format.

Coat 96-well plates with 1.0 µg/ml FMC63/0.1 M carbonate, pH 9.5 overnight at 4°C. The plate was blocked with 200 µl/well of Tris-buffered saline (TBS) containing 0.3% skim milk for 1 hour at room temperature. The plate was then washed three times with wash buffer (1× TBST: 0.1 M Tris, 0.5 M NaCl, 0.05% Tween 20). Next, 100 µl of #650 and #651 supernatants were serially diluted 3-fold starting at 5 µg/ml in TBS/1% BSA, pH 7.4 (dilution buffer). The samples were incubated at room temperature for 1 hour, and the plates were then washed as above. Next, 100 µl of dilution buffer containing 0.5 µg/ml biotinylated CD20 nanodiscs (ACROBiosystems) or biotinylated CD79b (homemade) was added to each well and incubated at 37°C for 1 hour. After washing the plate again, add 100 µl of 1:2000 diluted HRP-Streptavidin (in dilution buffer) to each well and incubate in the dark at 37°C for 1 hour. For detection, add 100 µl of 1-Step Ultra TMB-ELISA to each well. When color develops, add 100 µl of stop solution and read the plate at 450 nm. Graphs were generated using Prism software and are shown in Figures 23A and 23B.

As shown in Figure 23A, placing the CD79b scFv at the N- or C-terminal position of the anti-CD20 VHH did not significantly affect binding to biotinylated CD20. As shown in Figure 23B, binding to biotinylated CD79b does appear to be affected if the anti-CD79b scFv domain is located at the C-terminus of the molecule.

The ability to bind to cell surface CD20 or CD79b was examined by flow cytometric analysis. Controls binding to CD20 (#606 anti-CD20 VHH-CD19 ECD-anti-albumin) and CD79b (#645 anti-CD79b scFv-CD19 ECD) were included in this analysis. As shown in Figures 23C and 23D, #650 and #651 bind both antigens. Table 13 shows _EC50 values for binding to 293T-CD20 cells. Table 14 shows _EC50 values for binding to 293T-CD79b cells. Table 13: 293T-CD20 binding 606 645 650 651 EC50 (ng/mL) 6.70 - 34.45 24.46 EC50 (nM) 0.121 0.000 0.487 0.345 MW(Da) 55560 56800 70700 70800

CTE #650 and #651 have similar EC ₅₀ binding to CD20 expressed on the cell surface, but slightly lower than #606. 293T-CD20 binding 606 645 650 651 EC50 (ng/mL) - 425.80 200.9 269.4 EC50 (nM) 0.000 7.496 2.842 3.805 MW(Da) 55560 56800 70700 70800

Both #650 and #651 bind better than the monospecific anti-CD79b CTE #645 on cells expressing CD79b. These results indicate that anti-CD20 and anti-CD79b domains can bind their cognate antigens when present on the cell surface.

As shown in Figure 23E, CD20xCD79b CTE #650 and #651 were tested for cytotoxic activity against CD20+/CD79b+ JeKo-1 CD19 KO-luc cells. CTE3 was replaced with #606 protein or #645, #650 or #651 supernatant. Table 14 shows the _IC50 values for cytotoxicity against JeKo-1 CD19 KO cells. Table 14: IC ₅₀ ng/ml IC ₅₀ pM MW kDa #606 0.078 1.4 55.6 #645 10.45 184.0 56.8 #650 0.069 0.99 70.8 #651 0.037 0.52 70.8

Monospecific anti-CD20 CTE (#606) and bispecific anti-CD20/anti-CD79b CTE (#650/#651) can kill JeKo-1 CD19KO cells at an _IC50 value of approximately 1 pM. Anti-CD79b monospecific CTE has low potency. Example 14 : Surface plasmon resonance (SPR) results of CTE3 binding to anti -CD19 FMC63 and HSA

SPR measurements were performed on a Biacore 8K (Cytiva) at 25°C using 1× HBS-EP+ running buffer. In separate experiments for each ligand, biotinylated anti-CD19 and biotinylated HSA were immobilized to streptavidin-coated CM5 Biacore sensor chips. The biotin/streptavidin interaction is a high-affinity, non-covalent interaction in which the biotinylated substrate is almost irreversibly bound to the sensor chip surface. This coupling allows all immobilized molecules to be in the same orientation and high substrate densities can be achieved.

The CM5 wafer surface was activated by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS). Free biotin (0.2 μg/ml) was captured on flow cell (FC) 1: contact time 100 seconds, flow rate 10 μL/min. Anti-CD19 and HSA were diluted into running buffer (1.5 μg/mL and 4.0 μg/mL respectively) and injected onto FC2 in separate experiments until appropriate fixation levels were achieved (i.e. 1000-1500 responses Units (RU). Inactivate remaining streptavidin by injecting biotin (0.2 μg/ml, contact time 100 seconds, flow rate 10 μL/min) on all FCs.

For binding studies, serial dilutions of CTE3 (3.125, 6.25, 12.5, 25, 50, 100 and 200 nM) were prepared in running buffer and injected onto the immobilized target protein: association time 180 sec, dissociation time 900 sec, flow rate 30 μL/min. Subsequent wafer regeneration: regeneration buffer 10 mM glycine-HCl pH 1.5 for HSA binding, 10 mM glycine-HCl pH 2.0 for anti-CD19 binding, regeneration time for HSA binding is 30 seconds twice, anti-CD19 The combined regeneration time is every 30 seconds and the flow rate is 10 μL/min. In all experiments, free biotin immobilized on FC1 was used as a reference for blank subtraction.

As shown in Figures 24B and 24C, association and dissociation curves indicate that CTE3 has good affinity against CD19 and HSA, respectively. For anti-CD19, the equilibrium dissociation constant (K _D ) is 8.13 nM and the half-life (t½) is 15.03 minutes (Table 15). In a similar manner, the K _D of HSA was determined to be 65.1 nM with a t½ of 1.78 minutes (Table 15). Table 15 Ligand Ligand capture level (RU) k _a (1/Ms) k _d (1/s) K _D (nM) t _½ (min) Biotinylated HSA 156 9.92E+04 6.46E-03 65.1 1.78 Biotinylated anti- CD19 472 9.45E+04 7.68E-04 8.13 15.03 Example 15 : Surface plasmon resonance (SPR) results of CTE3 binding to CD20

SPR measurements were performed on a Biacore T200 (Cytiva) at 25°C using running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, 0.05% DDM, 0.01% CHS, pH7.4). Biotinylated CD20 was immobilized on a streptavidin-coated CM5 Biacore sensor chip. The biotin/streptavidin interaction is a high-affinity, non-covalent interaction in which the biotinylated substrate is almost irreversibly bound to the sensor chip surface. This coupling allows all immobilized molecules to be in the same orientation and high substrate densities can be achieved.

The CM5 wafer surface was activated by incubation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS). Free biotin (0.2 μg/ml) was captured on flow cell (FC) 1: contact time 100 seconds, flow rate 10 μL/min. Biotinylated CD20 was diluted into running buffer (0.2 μg/mL) and injected onto FC2 until appropriate fixation levels were achieved (i.e. 1000-1500 reaction units (RU). By injecting biotin on all FC (0.2 μg/ml, contact time 100 seconds, flow rate 10 μL/min) to inactivate remaining streptavidin.

For binding studies, use a desalting column to exchange CTE3 into running buffer. Serial dilutions of CTE3 (12.5, 25, 50, 100 and 200 nM) were prepared and injected onto the immobilized target protein: association 180 seconds, dissociation 2400 seconds, flow rate 30 μL/min. No regeneration step was performed. In all experiments, free biotin immobilized on FC1 was used as a reference for blank subtraction. All data analyzes were performed using Biacore evaluation software (Cytiva). The resulting association and dissociation curves fit the 1:1 binding model. Table 16 shows the measured kinetic parameters.

As shown in Figure 24A, association and dissociation curves indicate that CTE3 has good affinity for CD20 (K _D of 2.93 nM and t _½ of 52.23 minutes). The measured kinetic parameters for anti-CD20 are detailed in Table 16. Table 16 Ligand Ligand capture level (RU) k _a (1/Ms) k _d (1/s) K _D (nM) t _½ (min) Biotinylated CD20 200 7.56E+04 2.21E-04 2.93 52.23 Example 16 : CTE3 is more cytotoxic to cells expressing CD20 than CD20 x CD3 BiTE

This example further demonstrates that CTE3 mediates improved CAR-19 T cell cytotoxicity. Specifically, this example demonstrates that CTE3 is more cytotoxic to CD20-expressing cells than CD20 x CD3 BiTE after repeated stimulation of anti-CD19 CAR T cells

Raji cells (approximately 10 × 10 ⁶ in 1 ml) were treated with 0.5 µg/ml hygromycin C for 1 hour at 37°C to generate B cells for stimulation. Cells were washed twice with RPMI + 10% FBS (R10) and then resuspended at 1×10 ⁶ cells/ml. CAR19 cells were stimulated and restimulated with MMC-treated Raji every four days by co-culture at a ratio of 2 CAR19 cells:1 Raji cell. Cell density was maintained at approximately 0.5×10 ⁶ cells/ml. Every four days, just before restimulation, cells were sampled and stained with anti-CD3-FITC and anti-Flag-APC to determine CAR19 amplification; cell numbers were determined using flow cytometry with Bang Beads. After determining the CAR19 cell concentration, CTE3 or bispecific anti-CD20 x anti-CD3 (BPS Biosciences, catalog number: 100836-2) bridging cytotoxicity assay was performed on Jeko-1 CD19 KO cells using restimulated CAR19 cells. Read results 48 hours after analysis.

As shown in Figure 25A, the ability of CTE3 or anti-CD20 x anti-CD3 bispecific antibodies to kill Jeko-1 CD19 KO cells with CAR19 cells that had undergone one round of Raji cell stimulation was evaluated. The _IC50 of CTE3 is 2.8 pM and the _IC50 of the bispecific antibody is 35.7 pM. As shown in Figure 25B, the efficacy of CAR19 cells that had undergone 3 rounds of stimulation decreased. The IC ₅₀ of CTE3 (7.7 pM) is still lower than the IC ₅₀ of bispecific (124.1 pM).

These results indicate that CTE3 is more cytotoxic than a commercial anti-CD20 x anti-CD3 bispecific antibody when CAR19 cells are used in assays that have been repeatedly stimulated with B cells. Therefore, CTE3 may be more effective in preventing relapse in CAR19-treated patients. Example 17 : Batch Analysis of CTE3 Drug Products

This Example further demonstrates that compositions comprising fusion proteins described herein can be formulated to exhibit enhanced stability, reduced levels of aggregation and/or degradation, increased levels of protease resistance, increased antioxidant levels in vivo, Formulations with enhanced biodistribution and/or improved PK/PD properties.

A batch of CTE3 was produced and analyzed for various drug properties. Table 17 shows the detailed information and release testing of CTE3 batch. Table 17: Batch number 1A 1B Batch size/scale (small vials) 1000 3500 Production date June 24, 2022 August 9, 2022 Batch use Non-clinical, stability studies clinical, stability studies test method Specifications result result color Ph. Eur. ＜2.2.2＞ The color should not exceed No. 3 standard solution ＞Y7，＜Y6 (conforming) ＞UPW*，＜Y7 (conforming) Clarity Ph. Eur. ＜2.2.1＞ ≤ 18.0 NTU 3.0 NTU 2.0 NTU pH Ph. Eur. ＜2.2.3＞ USP＜791＞ 5.5-6.5 6.0 5.8 Osmotic weight molar concentration Ph. Eur. ＜2.2.35＞ USP＜785＞ 250 mOsmol/kg– 350 mOsmol/kg 293 mOsmol/kg 287 mOsmol/kg Visible particles Ph. Eur. ＜2.9.20＞ USP＜790＞ Liquid, essentially free of visible particles Liquid, no visible particles present Liquid, essentially free of visible particles subvisible particulate matter Ph. Eur. ＜2.9.19＞ USP＜787＞ ≥ 10 µm: ≤ 6000 particles/container ≥ 25 µm: ≤ 600 particles/container ≥ 5 µm: report the result; ≥ 2 µm: report the result. ≥ 2 μm: 869 particles/container; ≥ 5 μm: 222 particles/container; ≥ 10 μm: 26 particles/container; ≥ 25 μm: 0 particles/container ≥ 2 μm: 2198 particles/container; ≥ 5 μm: 449 particles/container; ≥ 10 μm: 62 particles/container; ≥ 25 μm: 2 particles/container Extractable volume Ph. Eur. ＜2.9.17＞ USP＜1＞ ≥ 15.0mL ＞15.0mL ＞15.0mL protein concentration UV 9.0 mg/mL–11.0 mg/mL 9.9 mg/mL 10.1 mg/mL Desialylated iCIEF Desialylated iCIEF Comply with reference standards Comply with reference standards Comply with reference standards Desialylated iCIEF Desialylated iCIEF Main peak %: ≥ 35.0% Acidic peak %: ≤ 65.0% Basic peak %: ≤ 10.0% Main peak %: 55.5%; Acidic peak %: 44.1%; Basic peak %: 0.4% Main peak %: 51.5%; Acidic peak %: 47.8%; Basic peak %: 0.8% iCIEF iCIEF Group A: ≥ 90.0% Group A: 100.0% Group A: 100.0% UPLC-SEC UPLC-SEC Monomer% (main peak%): ≥ 95.0%; HMWS%: ≤5.0% Monomer% (main peak%): 99.3%; HMWS%: 0.7% Monomer% (main peak%): 99.3%; HMWS%: 0.7% Deglycosylated and reduced CE-SDS Deglycosylated and reduced CE-SDS Purity%: ≥ 90.0% LMWS%: ≤ 10.0% Purity%: 96.7%; LMWS%: 3.3% Purity%: 95.7%; LMWS%: 4.3% ELISA binding (anti-CD19 and CD20) ELISA 50% - 150% relative potency 104% relative efficacy 100% ELISA binding (anti-CD19 and HSA) ELISA 50% - 150% relative potency 116% relative efficacy 97% sterility Ph. Eur. ＜2.6.1＞ USP＜71＞ No growth No growth No growth bacterial endotoxin Ph. Eur. ＜2.6.14＞ USP＜85＞ ≤ 12.50 EU/mL 2.00 EU/mL ＜0.20 EU/mL Polysorbate 80 content Polysorbate 80 0.010% – 0.030% 0.021% 0.019% CCIT USP＜1207＞ Not applicable NA NA

The stability of test batch 1B was evaluated after 1 month and 3 months. Table 18 shows the immediate stability data for test batch 1B. Table 18 Acceptance criteria T=0 T=1 month T= March The color should not exceed No. 3 standard solution ＞UPW，＜Y7 (conforming) ＞UPW，＜Y7 (conforming) ＞UPW，＜Y7 (conforming) ≤ 18.0 NTU 2.0 NTU 1.8 NTU 1.9 NTU 5.5 – 6.5 5.8 6.0 6.0 Liquid, essentially free of visible particles Liquid, essentially free of visible particles Liquid, essentially free of visible particles Liquid, essentially free of visible particles ≥10 µm: ≤ 6000 particles/container ≥25 µm: ≤ 600 particles/container ≥5 µm: report the result; ≥2 µm: report the result. ≥ 2 μm: 2198 particles/container; ≥ 5 μm: 449 particles/container; ≥ 10 μm: 62 particles/container; ≥ 25 μm: 2 particles/container ≥ 2 μm: 1887 particles/container; ≥ 5 μm: 300 particles/container; ≥ 10 μm: 29 particles/container; ≥ 25 μm: 2 particles/container ≥ 2 μm: 2331 particles/container; ≥ 5 μm: 497 particles/container; ≥ 10 μm: 68 particles/container; ≥ 25 μm: 0 particles/container 9.0 mg/mL–11.0 mg/mL 10.1 mg/mL 9.9 mg/mL 10.1 mg/mL Main peak %: ≥ 35.0%; Acidic peak %: ≤ 65.0%; Basic peak %: ≤ 10.0% Main peak %: 51.5%; Acidic peak %: 47.8%; Basic peak %: 0.8% Main peak %: 53.7%; Acidic peak %: 45.9%; Basic peak %: 0.4% Main peak %: 53.8%; Acidic peak %: 45.7%; Basic peak %: 0.4% Group A%: ≥ 90.0% Group A: 100.0% NT NT Monomer% (main peak%): ≥95.0%; HMWS%: ≤ 5.0% Monomer% (main peak%): 99.3%; HMWS%: 0.7% Monomer% (main peak%): 99.2%; HMWS%: 0.8% Monomer% (main peak%): 99.3%; HMWS%: 0.7% Purity%: ≥ 90.0%; LMWS%: ≤ 10.0% Purity%: 95.7%; LMWS%: 4.3% Purity%: 96.8%; LMWS%: 3.2% Purity%: 96.4%; LMWS%: 3.6% 0.010% – 0.030% 0.019% NT NT 50% - 150% relative potency 100% 101% 99% 50% - 150% relative potency 97% 100% 102% qualified NT NT NT

As shown in Table 18, after 1 month and 3 months, the compositions containing CTE3 exhibited similar properties as when initially tested.

In another example, the stability of compositions containing CTE3 over a time point of more than 3 months was evaluated. Table 19 describes stability storage conditions and testing time points. Compositions containing CTE3 remain stable for at least 3 months. Table 19: Batch number stability type Storage conditions time point 20220601 Instant -20±5℃ T0, 1M, 3M, 6M, 9M, 12M, 18M, 24M, 30M, 36M, 42M, 48M, 60M accelerate 5±3℃ T0, 1M, 3M, 6M pressure 25 ± 2℃/ 60 ± 5%RH T0, 1M, 2M, 3M 20220802 Instant -20±5℃ T0, 1M, 3M, 6M, 9M, 12M, 18M, 24M, 36M, 48M, 60M accelerate 5±3℃ T0, 1M, 3M, 6M pressure 25 ± 2℃/ 60 ± 5%RH T0, 1M, 2M, 3M equivalent

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but is as stated in the following patent application scope: Sequence Listing SEQ ID NO: 1 SEQ ID NO: 2 (the order is: [signal sequence]-[VHH ( bold font indicates CDR1 , CDR2 , CDR3 )]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Hexahistidine tag]) [MGWSCIILFLVATATGVHS]- [ QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDW NAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]- [HHHHHH] SEQ ID NO:3 SEQ ID NO:4 (順序為：[VHH(粗體表示 CDR1 、 CDR2 、 CDR3 )]- [第一連接子]-[目標多肽]-[第二連接子]-[半衰期延長多肽]) [ QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:5 SEQ ID NO:6 (The order is: [signal sequence]-[VHH ( bold indicates CDR1 , CDR2 , CDR3 )]-[first linker]-[target polypeptide]-[second linker ]-[半衰期延長多肽]-[六組胺酸標籤]) [MEFGLSWVFLVALFRGVQC]- [ QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH] SEQ ID NO:7 SEQ ID NO:8 (The sequence is: [VHH ( bold indicates CDR1 , CDR2 , CDR3 )]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide ]) [ QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:9 SEQ ID NO:10 (順序為：[信號序列]-[VHH(粗The body represents CDR1 , CDR2 , CDR3 )]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Hexahistidine acid tag]) [MEFGLSWVFLVALFRGVQC]- [ QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ISWSGGWI YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]- [HHHHHH] SEQ ID NO:11 SEQ ID NO:12 (順序為：[VHH(粗體表示 CDR1 、 CDR2 、 CDR3 )]-[第一連接子]-[目標多肽]-[第二連接子]-[半衰期延長多肽]) [ QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ISWSGGWI YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS] -[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO: 13 SEQ ID NO: 14 (The sequence is: [signal sequence]-[VHH ( bold indicates CDR1 , CDR2 , CDR3 )]-[first linker]-[target polypeptide]- [第二連接子]-[半衰期延長多肽]-[六組胺酸標籤]) [MEFGLSWVFLVALFRGVQC]- [ QCQVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ISWSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]- [HHHHHH] SEQ ID NO:15 SEQ ID NO:16 (The order is: [VHH ( bold indicates CDR1 , CDR2 , CDR3 )]-[first linker]-[target polypeptide]-[second linker]- [半衰期延長多肽]) [ QCQVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ISWSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]- [PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP][SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:17 SEQ ID NO:18 (順序為：[信號序列]-[ VHH ( bold indicates CDR1 , CDR2 , CDR3 )]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Hexahistidine acid tag]) [MEFGLSWVFLVALFRGVQC]- [ QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGWI YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH] SEQ ID NO:19 SEQ ID NO:20 (順序為：[VHH(粗體表示 CDR1 、 CDR2 、 CDR3 )]-[第一連接子]-[目標多肽]-[第二連接子]-[半衰期延長多肽]) [ QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGWI YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]- [SRGGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:21 SEQ ID NO:22 (The sequence is: [signal sequence]-[VHH ( bold indicates CDR1 , CDR2 , CDR3 )]-[ First linker]-[target polypeptide ]-[第二連接子]-[半衰期延長多肽]-[六組胺酸標籤]) [MEFGLSWVFLVALFRGVQC]-[QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ISWSGGST YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[ EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH] SEQ ID NO: 23 SEQ ID NO: 24 (The order is: [VHH ( bold indicates CDR1 , CDR2 , CDR3 )]-[First linker]-[Target polypeptide]-[ Second connection子]-[半衰期延長多肽]) [QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ISWSGGST YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:25 SEQ ID NO:26 (順序為：[信號Sequence]-[VHH ( Bold indicates CDR1 , CDR2 , CDR3 )]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Hexahistidine acid tag]) [MEFGLSWVFLVALFRGVQC]-[QCQVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGST YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH] SEQ ID NO:27 SEQ ID NO:28 (順序為：[VHH( Bold indicates CDR1 , CDR2 , CDR3 )]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]) [QCQVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGST YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS ]-[GGGGSGGGGSGGGGSGGGGS]- [PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:29 SEQ ID NO:30 (順序為：[信號序列]-[VHH(粗體表示 CDR1 、 CDR2 、 CDR3 )]-[第一連接子] -[目標多肽]-[第二連接子]-[半衰期延長多肽]-[六組胺酸標籤]) [MEFGLSWVFLVALFRGVQC]-[QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AADPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[ SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH] SEQ ID NO:31 SEQ ID NO:32 (The order is: [VHH ( bold indicates CDR1 , CDR2 , CDR3 )]-[ First linker]-[Target polypeptide]- [第二連接子]-[半衰期延長多肽]) [QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AADPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:33 SEQ ID NO:34 (順序It is: [Signal sequence]-[VHH ( bold indicates CDR1 , CDR2 , CDR3 )]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]-[Hexahistamine酸標籤]) [MEFGLSWVFLVALFRGVQC]-[QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGWI YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AADPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH] SEQ ID NO:35 SEQ ID NO:36 (順序為: [VHH ( Bold indicates CDR1 , CDR2 , CDR3 )]-[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension polypeptide]) [QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGWI YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AADPTYGSDWNAEN WGQGTQ VTVSS]- [GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:37 (粗體表示 CDR1 、 CDR2 、 CDR3 ) QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS SEQ ID NO:38 (粗體表示 CDR1 、 CDR2 、 CDR3 ) QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ISWSGGWI YYASSVRGRFTISSRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS SEQ ID NO:39 ( bold indicates CDR1 , CDR2 , CDR3 ) QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFR QAPGKEREFVAA ISWSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS SEQ ID NO:40 ( bold indicates CDR1 , CDR2 , CDR3 ) QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGWI YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS SEQ ID NO:41 ( Bold indicates CDR1 , CDR2 , CDR3 ) QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ISWSGGST YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANP TYGSDWNAEN WGQGTQVTVSS SEQ ID NO:42 ( Bold indicates CDR1 , CDR2 , CDR3 ) QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGST YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS SEQ ID NO :43 ( Bold indicates CDR1 , CDR2 , CDR3 ) QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AADPTYGSDWNAEN WGQGTQVTVSS SEQ ID NO:44 ( Bold indicates CDR1 , CDR2 , CDR3 ) QVQL QESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGWI YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AADPTYGSDWNAEN WGQGTQVTVSS SEQ ID NO:45Anti -albumin VHH EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS SEQ ID NO:46 PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP SEQ ID NO:47 SEQ ID NO:48 PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARP SEQ ID NO:49 SEQ ID NO:50 (順序為：[信號序列]-[VHH(粗體表示 CDR1 、 CDR2 、 CDR3 )] -[First linker]-[Target polypeptide]-[Second linker]-[Half-life extension peptide]-[Hexahistidine tag]) [MEFGLSWVFLVALFRGVQC]-[EVQLVESGGGLVQPGGSLRLSCTFS GGTFSSYT MGWFRQAPGKEREFVAE VRWGGVT TYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYC AAVRQMYMTVVPDY WGQGTLVTV SS]-[GGGGSGGGGSGGGGSGGGGS ]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS]-[HHHHHH] SEQ ID NO:51 SEQ ID NO:52 (順序為：[VHH(粗體表示 CDR1 、 CDR2 、 CDR3 )]-[第一連接子]-[目標多肽]-[第二連接子]-[半衰期延長多肽]) [EVQLVESGGGLVQPGGSLRLSCTFS GGTFSSYT MGWFRQAPGKEREFVAE VRWGGVT TYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYC AAVRQMYMTVVPDY WGQGTLVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS]-[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:53 ( Bold indicates CDR1 , CDR2 , CDR3 ) EVQLVESGGGLVQPGGSLRLSCTFS GGTFSSYT MGWFRQAPGKEREFVAE VRWGGVT TYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYC AAVRQMYMTVVPDY WGQGTLVTVSS SEQ ID NO:54 SEQ ID NO:55 (The sequence is: [Signal sequence]-[VHH ( Bold indicates CDR1 , CDR2 , CDR3 )]- [第一連接子]-[目標多肽]-[第二連接子]-[半衰期延長多肽]) [MGWSCIILFLVATATGVHS]- [ QVQLQESGGGLAQAGGSLRLSCAAS GRTFS MGWFRQAPGKEREFVAA ITYSGGSP YYASSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AANPTYGSDWNAEN WGQGTQVTVSS]-[GGGGSGGGGSGGGGSGGGGS]-[PEEPLVVKVEEGDTAALWCLKGTSDGPTQQLTWSRESPLKPFLKYSLGVPGLGVHVRPDAISVVIRNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDLTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMGTSLMLPRATAQDAGKWYCHRGNLTMSFHLEITARP]-[SRGGGGSGGGGSGGGGS] -[EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS] SEQ ID NO:56 PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEK AWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKSE

without

Numbers in the figures are for illustrative purposes only and are not intended to be limiting.

Figure 1 shows the isoelectric point distribution of protein preparations CTE1, CTE2 and CTE3. CTE1 is a mixture of sialylated and non-sialylated proteins, CTE2 is mainly non-sialylated, and CTE3 is mainly sialylated.

Figure 2 shows the isoelectric point distribution of the protein preparation CTE1 after various treatments to remove sialylation (eg, sialidase).

Figure 3 shows the binding affinity curves of the protein preparations CTE1 (red circles), CTE2 (green squares) and CTE3 (blue circles) to CD19-negative/CD20-positive JeKo-1 cells (JeKo-19KO), as analyzed by flow cytometry Measured by technology.

Figure 4 shows the protein preparations CTE1 (green triangle), CTE2 (red circle) and CTE3 (blue square) in vivo with purified biotinylated human albumin (left panel) or biotinylated human CD20 membrane preparation (right panel) External binding affinity curve as measured by ELISA.

Figure 5 shows the effects of the protein formulations CTE1 (red circles), CTE2 (blue squares), and CTE3 (pink triangles) on CD19-negative/CD20-positive JeKo-1 cells (JeKo-19KO) when administered with CAR-19 T cells. Kill curve.

Figure 6 shows that wild-type JeKo-1 cells are predominantly positive for CD19 and CD20, as measured by flow cytometric analysis.

Figures 7A-7C show CD19 and CD20 expression levels of JeKo-1 B-cell lymphoma cells treated with CAR-19 T cells over 13 days, as measured by flow cytometric analysis. Figures 7A, 7B, and 7C correspond to experiments in which the CAR-19 T:JeKo-1 cell ratio was 1:1 (A), 0.3:1 (B), or 0.1:1 (C).

Figure 8A shows CD19 and CD20 expression levels of CAR-19 T cells and JeKo-1 cells cultured at a 1:1 ratio for 13 days, as measured by flow cytometric analysis. Figure 8B shows the corresponding flow cytometric analysis of subsequent addition of CAR-19 T cells, fusion protein (middle panel), or both (bottom) to cells.

Figure 9 shows the time course of bioluminescence imaging of mice injected with JeKo-19KO lymphoma cells and treated with CAR-19 T cells plus CTE1, CAR-19 T cells alone, CAR-20 T cells alone, or untreated. .

Figure 10 shows the time course of bioluminescence imaging of mice injected with JeKo-19KO lymphoma cells and treated with CAR-19 T cells plus CTE1, CAR-19 T cells plus CTE2, CAR-19 T cells alone, or untreated. .

Figure 11 shows the time course of bioluminescence imaging of mice injected with JeKo-19KO lymphoma cells and treated with different concentrations of CAR-19 T cells plus CTE2, CAR-19 T cells alone, or untreated.

Figure 12 shows the average body weight of the mice reported in Figure 11.

Figure 13 shows the average luminescence of the mice reported in Figure 11. The picture on the right is an expanded view of 0.016, 0.08, 0.4 and 2 mg/kg fusion proteins.

Figure 14 shows the survival probability of the mice reported in Figure 11.

Figure 15 shows the time course of the protein concentration of the fusion proteins CTE1, CTE2 and CTE3 in the serum of NSG mice, which is used to estimate the half-life of the protein in the circulation in vivo.

Figure 16 shows the binding affinity curves of various fusion protein constructs to CD20 positive/CD19 negative JeKo-1 cells (JeKo-19KO) as measured by ELISA.

Figure 17 shows the killing curves of various fusion protein constructs against CD19 negative/CD20 positive JeKo-1 cells (JeKo-19KO) when administered with CAR-19 T cells.

Figure 18 shows binding and killing curves for additional fusion proteins.

Figure 19A shows the binding affinity curve of CTE3 fusion protein to CD19-negative/CD20-positive JeKo-1 cells (JeKo-19KO), as measured by flow cytometric analysis. Figure 19B shows the binding affinity curve of CTE3 fusion protein to wild-type JeKo-1 cells, as measured by flow cytometric analysis. Figure 19C shows the binding affinity curve of the fusion protein to wild-type Ramos-Hodgkin's lymphoma cells, as measured by flow cytometric analysis.

Figure 20 shows the killing curve of CTE3 fusion protein formulations against CD19 negative/CD20 positive JeKo-1 cells (JeKo-19KO) when administered with CAR-19 T cells.

Figure 21A shows binding of CTE3 to 293T cells transfected with cynomolgus CD20. Figure 21B shows binding of CTE3 to 293T cells transfected with human CD20.

Figure 22A shows the effect of human serum on CTE3-mediated CAR19 cytotoxicity. Increased CTE3 levels are shown on the x-axis. HS=human serum. Figure 22B shows the effect of human serum albumin (HSA) on CTE3-mediated CAR19 cytotoxicity. Increased CTE3 levels are shown on the x-axis.

Figure 23A shows that CD79b x CD20 bispecific CTE binds biotinylated CD20. Figure 23B shows CTE #650 binds biotinylated CD79b. Figure 23C shows binding of monospecific and bispecific anti-CD20 and anti-CD79b proteins to 293T-CD20 cells. Figure 23D shows binding of monospecific and bispecific anti-CD20 and anti-CD79b proteins to 293T-CD79b cells. Figure 23E shows the cytotoxic ability of anti-CD19 CAR T cells plus monospecific and bispecific anti-CD20 and anti-CD79b proteins against JeKo-1 CD19KO cells.

Figure 24A shows binding of CTE3 to biotinylated CD20 using SPR. Figure 24B shows binding of CTE3 to biotinylated CD19 using SPR. Figure 24C shows binding of CTE3 to biotinylated HSA using SPR.

Figure 25A shows the cytotoxicity curve of JeKo-1 CD19 KO cells after one round of CAR19 stimulation. Figure 25B shows the cytotoxicity curve of JeKo-1 CD19 KO cells after three rounds of CAR19 stimulation.

without

Claims (51)

A fusion protein comprising (i) an antibody that binds a tumor antigen or an antigen-binding fragment thereof; (ii) a target polypeptide; and (iii) a half-life extending polypeptide. Such as the fusion protein of claim 1, wherein the half-life extending polypeptide is any one of the following: hyaluronic acid binding motif, PAS polypeptide, proline/alanine random coil polypeptide and antibody or antigen-binding fragment thereof. The fusion protein of claim 2, wherein the half-life extending polypeptide is an antibody or an antigen-binding fragment thereof. Such as the fusion protein of claim 3, wherein the half-life extending polypeptide is an anti-albumin antibody or an antigen-binding fragment thereof. The fusion protein of claim 4, wherein the anti-albumin antibody or antigen-binding fragment thereof includes anti-albumin VHH. The fusion protein of claim 5, wherein the anti-albumin VHH comprises an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical to SEQ ID NO: 45. The fusion protein of any one of claims 1-6, wherein the tumor antigen is CD20, and wherein the antibody or antigen-binding fragment thereof is an anti-CD20 antibody or antigen-binding fragment thereof. The fusion protein of claim 7, wherein the anti-CD20 antibody or antigen-binding fragment thereof includes anti-CD20 VHH. The fusion protein of claim 8, wherein the anti-CD20 VHH comprises the amino acid sequence of any one of SEQ ID NOs: 37, 39, 40, 42-44 and 53. The fusion protein of claim 8, wherein the anti-CD20 VHH comprises the amino acid sequence of CDR1, CDR2 or CDR3 described in any one of SEQ ID NO: 37, 39, 40, 42-44 and 53. The fusion protein of any one of claims 1-10, wherein the target polypeptide is a tumor antigen. The fusion protein of any one of claims 1-11, wherein the target polypeptide is a B cell antigen. Such as the fusion protein of any one of claims 1-12, wherein the target polypeptide is CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, CD180, ROR1, BCMA, HLA-DR10, CD1, CD5, CD21 , any of CD25, CD27, CD30, CD38, CD78, CD80, CD86, CD138, CD319, surface Ig, PD-1, PD-L1, PD-L2, TGFbR2, CD79a and CD79b. The fusion protein of any one of claims 1-13, wherein the target polypeptide is CD19 or a fragment or mutant thereof. The fusion protein of claim 14, wherein the CD19 comprises an amino acid sequence that is at least about 95%, at least about 97%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 47. The fusion protein of claim 14, wherein the target polypeptide comprises about 230, about 240, about 250, about 260 or about 270 consecutive amino acids of SEQ ID NO: 47. The fusion protein of claim 14, wherein the target polypeptide includes amino acids 20-278 of SEQ ID NO: 47. The fusion protein of claim 14, wherein the target polypeptide comprises an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino acid sequence of SEQ ID NO: 48 or 56. The fusion protein of claim 14, wherein the target polypeptide comprises an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino sequence of SEQ ID NO: 46. The fusion protein of claim 14, wherein the target polypeptide includes the amino acid sequence of SEQ ID NO: 46. Such as the fusion protein of any one of claims 1-20, the fusion protein includes the first linker between the antibody or its antigen-binding fragment and the target polypeptide. Such as the fusion protein of any one of claims 1-21, the fusion protein includes a second linker between the target polypeptide and the half-life extending polypeptide. The fusion protein of claim 21 or 22, wherein the first linker or the second linker includes the amino acid sequence of any one of the following (i) GS linker, (ii) polyglycine linker, (iii) linkers rich in glycine and serine, or (iv) Flexible connectors. The fusion protein of claim 21, wherein the first linker includes the amino acid sequence GGGGSGGGGSGGGGSGGGGS. The fusion protein of claim 22, wherein the second linker includes the amino acid sequence SRGGGGSGGGGSGGGGS. The fusion protein of any one of claims 1-25, wherein the fusion protein contains SEQ ID NO: 2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, The amino acid sequence of any one of 36, 50, 52, and 55 has an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical. The fusion protein of any one of claims 1-26, wherein the fusion protein includes SEQ ID NO: 2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36 The amino acid sequence of any one of , 50, 52 and 55. The fusion protein of any one of claims 1-27, wherein the fusion protein consists of SEQ ID NO: 2, 4, 6, 8, 14, 16, 18, 20, 26, 28, 30, 32, 34, 36 , 50, 52 and 55 any one of the amino acid sequence composition. The fusion protein of any one of claims 1-26, wherein the fusion protein includes an amino acid sequence identical to any one of SEQ ID NO: 2, 6, 14, 18, 26, 30, 34 and 50. An amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical and lacks a C-terminal hexahistidine tag. The fusion protein of any one of claims 1-26, wherein the fusion protein includes the amino acid sequence of any one of SEQ ID NO: 2, 6, 14, 18, 26, 30, 34 and 50, and Missing C-terminal hexahistidine tag. Such as the fusion protein of any one of claims 1-26, wherein the fusion protein consists of the amino acid sequence of any one of SEQ ID NO: 2, 6, 14, 18, 26, 30, 34 and 50, and lacks the C-terminal hexahistidine tag. Such as the fusion protein of any one of claims 1-31, the fusion protein includes a second antibody or an antigen-binding fragment thereof that binds a second tumor antigen. Such as the fusion protein of claim 32, wherein the second tumor antigen is CD79b, HER-2/neu, c-met, EGFR, Ga733\EpCAM, CD21, ROR1, CLL-1/CLEC12A or BCMA. A nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of the fusion protein of any one of claims 1-33. A nucleic acid comprising (i) any one of SEQ ID NOs: 1, 3, 5, 7, 13, 15, 17, 19, 25, 27, 29, 31, 33, 35, 49, 51 and 54 The nucleotide sequence, or (ii) the nucleotide sequence of any one of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33 and 49, and lacking the coding for hexahistamine The last 18 nucleotides of the acid tag. A vector comprising the nucleic acid of claim 34 or 35. A host cell comprising the nucleic acid of claim 34 or 35 or the vector of claim 28. A method of producing a fusion protein, comprising culturing the host cell of claim 34 under conditions suitable for expressing the fusion protein. A method of treating an individual suffering from a tumor, comprising administering to the individual an effective amount of the fusion protein of any one of claims 1-33, thereby treating the individual. The method of claim 39, wherein the tumor expresses a tumor antigen. Claim the method of item 39 or 40, wherein the tumor does not express CD19. The method of any one of claims 39-41, wherein after administration, the fusion protein binds to the tumor antigen. The method of any one of claims 39-42, further comprising administering to the individual an antibody, antibody-drug conjugate, or cell therapy (eg, CAR-T cell) that specifically recognizes the target polypeptide. The method of claim 43, wherein the antibody, antibody drug conjugate or cell therapy (eg, CAR-T cell) binds to the fusion protein after administration to the individual. The method of claim 44, wherein binding of the antibody, the antibody-drug conjugate, or the cell therapeutic agent (eg, CAR-T cells) to the fusion protein induces killing of the tumor. An antibody or an antigen-binding fragment thereof, comprising a VHH or a fragment thereof having the amino acid sequence of any one of SEQ ID NO: 37, 39, 40, 42-44 and 53. An antibody or antigen-binding fragment thereof, wherein the VHH comprises at least one CDR (e.g., CDR1, CDR2 and/or CDR3) described in any one of SEQ ID NOs 37, 39, 40, 42-44 and 53. A nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 46 or 47. A vector comprising the nucleic acid sequence of claim 48. A host cell comprising the nucleic acid sequence of claim 48 or the vector of claim 49. A method of producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell of claim 50 under conditions suitable for expressing the antibody or an antigen-binding fragment thereof or a fusion protein.